Isolated BOS1 gene promoters from arabidopsis and uses thereof

ABSTRACT

The present invention pertains to an isolated promoter sequence from  Arabidopisis thaliana  BOSI gene encoding a protein for biotic and abiotic stress tolerance. Also, the invention relates to recombinant vectors, expression cassettes, host cells, plants or progeny thereof comprising nucleic acid molecules operably linked to said promoter.

This application is a § 371 of International Application No.PCT/US03/07331, filed Mar. 5, 2003, which claims the benefit of U.S.Provisional Patent Application No. 60/361,861 filed Mar. 5, 2002, whichis incorporated herein by reference.

FIELD OF THE INVENTION

The present invention pertains to nucleic acid molecules isolated fromArabidopsis thaliana comprising nucleotide sequences that encodeproteins for biotic and abiotic stress tolerance. The inventionparticularly relates to methods of using nucleic acid molecules and/orproteins from Arabidopsis in transgenic plants to confer enhanced bioticand/or abiotic stress tolerance, and to use such nucleic acids to assistgermplasm enhancement by breeding. In particular, the invention pertainsto nucleic acid molecules isolated from Arabidopsis thaliana of the BOS1cDNA and genomic gene and encoding the BOS1 polypeptide. The presentinvention also pertains to nucleic acid molecules isolated fromArabidopsis thaliana that encode a promoter from the BOS1 gene, andmethods of using the promoter. Also, the invention relates to hostcells, plants or progeny thereof comprising the nucleic acid moleculesor recombinant molecules described herein.

BACKGROUND OF THE INVENTION

Improvement of the agronomic characteristics of crop plants has beenongoing since the beginning of agriculture. Most of the land suitablefor crop production is currently being used. As human populationscontinue to increase, improved crop varieties will be required toadequately provide our food and feed (Trewavas (2001) Plant Physiol.125: 174–179). To avoid catastrophic famines and malnutrition, futurecrop cultivars will need to have improved yields with equivalent farminputs. These cultivars will need to more effectively withstand adverseconditions such as drought, soil salinity or disease, which will beespecially important as marginal lands are brought into cultivation.Finally, we will need cultivars with altered nutrient composition toenhance human and animal nutrition, and to enable more efficient foodand feed processing, by designing cultivars for specific end-uses. Forall these traits, identification of the genes controlling phenotypicexpression of traits of interest will be crucial in acceleratingdevelopment of superior crop germplasm by conventional or transgenicmeans.

A number of highly efficient approaches are available to assistidentification of genes playing key roles in expression of agronomicallyimportant traits. These include genetics, genomics, bioinformatics, andfunctional genomics. Genetics is the scientific study of the mechanismsof inheritance. By identifying mutations that alter the pathway orresponse of interest, classical (or forward) genetics can help toidentify the genes involved in these pathways or responses. For example,a mutant with enhanced susceptibility to disease may identify animportant component of the plant signal transduction pathway leadingfrom pathogen recognition to disease resistance. Genetics is also thecentral component in improvement of germplasm by breeding. Throughmolecular and phenotypic analysis of genetic crosses, loci controllingtraits of interest can be mapped and followed in subsequent generations.Knowledge of the genes underlying phenotypic variation between cropaccessions can enable development of markers that greatly increaseefficiency of the germplasm improvement process, as well as open avenuesfor discovery of additional superior alleles. Genomics is thesystem-level study of an organism's genome, including genes andcorresponding gene products—RNA and proteins. At a first level, genomicapproaches have provided large datasets of sequence information fromdiverse plant species, including full-length and partial cDNA sequences,and the complete genomic sequence of a model plant species, Arabidopsisthaliana. Recently, the first draft sequence of a crop plant's genome,that of rice (Oryza sativa), has also become available. Availability ofwhole genome sequence makes possible the development of tools forsystem-level study of other molecular complements, such as arrays andchips for use in determining the complement of expressed genes in anorganism under specific conditions. Such data can be used as a firstindication of the potential for certain genes to play key roles inexpression of different plant phenotypes. Bioinformatics approachesinterface directly with first-level genomic datasets in allowing forprocessing to uncover sequences of interest by annotative or othermeans. Using, for example, similarity searches, alignments andphylogenetic analyses, bioinformatics can often identify homologs of agene product of interest. Very similar homologs (eg. >˜90% amino acididentity over the entire length of the protein) are very likelyorthologs, i.e. share the same function in different organisms.

Functional genomics can be defined as the assignment of function togenes and their products. Functional genomics draws from genetics,genomics and bioinformatics to derive a path toward identifying genesimportant in a particular pathway or response of interest. Expressionanalysis, for example, uses high density DNA microarrays (often derivedfrom genomic-scale organismal sequencing) to monitor the mRNA expressionof thousands of genes in a single experiment. Experimental treatmentscan include those eliciting a response of interest, such as the diseaseresistance response in plants infected with a pathogen. To giveadditional examples of the use of microarrays, mRNA expression levelscan be monitored in distinct tissues over a developmental time course,or in mutants affected in a response of interest. Proteomics can alsohelp to assign function, by assaying the expression andpost-translational modifications of hundreds of proteins in a singleexperiment. Proteomics approaches are in many cases analogous to theapproaches taken for monitoring mRNA expression in microarrayexperiments. Protein-protein interactions can also help to assignproteins to a given pathway or response, by identifying proteins whichinteract with known components of the pathway or response. Forfunctional genomics, protein-protein interactions are often studiedusing large-scale yeast two-hybrid assays. Another approach to assigninggene function is to express the corresponding protein in a heterologoushost, for example the bacterium Escherichia coli, followed bypurification and enzymatic assays.

Ultimately, demonstration of the ability of a gene-of-interest tocontrol a given trait must be derived from experimental testing in plantspecies of interest. The generation and analysis of plants transgenicfor a gene of interest can be used for plant functional genomics, withseveral advantages. The gene can often be both overexpressed andunderexpressed (“knocked out”), thereby increasing the chances ofobserving a phenotype linking the gene to a pathway or response ofinterest. Two aspects of transgenic functional genomics help lend a highlevel of confidence to functional assignment by this approach. First,phenotypic observations are carried out in the context of the livingplant. Second, the range of phenotypes observed can be checked andcorrelated with with observed expression levels of the introducedtransgene. Transgenic functional genomics is especially valuable inimproved cultivar development. Only genes that function in a pathway orresponse of interest, and that in addition are able to confer a desiredtrait-based phenotype, are promoted as candidate genes for cropimprovement efforts. In some cases, transgenic lines developed forfunctional genomics studies can be directly utilized in initial stagesof product development.

Another approach towards plant functional genomics involves firstidentifying plant lines with mutations in specific genes of interest,followed by phenotypic evaluation of the consequences of such geneknockouts on the trait under study. Such an approach reveals genesessential for expression of specific traits.

Genes identified through functional genomics can be directly employed inefforts towards germplasm improvement by transgenic means, as describedabove, or used to develop markers for identification of tracking ofalleles-of-interest in mapping and breeding populations. Knowledge ofsuch genes may also enable construction of superior alleles non-existentin nature, by any of a number of molecular methods.

SUMMARY OF THE INVENTION

This Summary of Invention lists several embodiments of the invention,and in many cases lists variations and permutations of theseembodiments. This Summary is merely exemplary of the numerous and variedembodiments. Mention of one or more preferred features of a givenembodiment is likewise exemplary. Such embodiment can typically existwith or without the feature(s) mentioned; likewise, those features canbe applied to other embodiments of the invention, whether listed in thisSummary or not. To avoid excessive repetition, this Summary does notlist or suggest all possible combinations of such features.

Embodiments of the present invention provide nucleotide and amino acidsequences known as cDNAs from rice.

Embodiments of the present invention relate to an isolated nucleic acidcomprising or consisting of a nucleotide sequence including:

-   -   (a) a nucleotide sequence listed in SEQ ID NO:1, fragment,        domain, or feature thereof;    -   (b) a nucleotide sequence having substantial similarity to (a);    -   (c) a nucleotide sequence capable of hybridizing to (a);    -   (d) a nucleotide sequence complementary to (a), (b) or (c); and    -   (e) a nucleotide sequence which is the reverse complement of        (a), (b) or (c).

In a preferred embodiment, the substantial similarity is at least about65% identity, preferably about 80% identity, preferably 90%, and morepreferably at least about 95% identity to the nucleotide sequence listedin SEQ ID NO:1, fragment, domain, or feature thereof.

In a preferred embodiment, the sequence having substantial similarity tothe nucleotide sequence listed in SEQ ID NO:1, fragment, domain, orfeature thereof, is from a plant. In a preferred embodiment, the plantis a dicot. In another preferred embodiment, the plant is a gymnosperm.In a more preferred embodiment, the plant is a monocot. In a morepreferred embodiment, the monocot is a cereal. In a more preferredembodiment, the cereal may be, for example, maize, wheat, barley, oats,rye, millet, sorghum, triticale, secale, einkorn, spelt, emmer, teff,milo, flax, gramma grass, Tripsacum sp., or teosinte. In a mostpreferred embodiment, the cereal is rice.

In a preferred embodiment, the nucleic acid is expressed in a specificlocation or tissue of a plant. In a more preferred embodiment, thelocation or tissue is for example, but not limited to, epidermis, root,vascular tissue, meristem, cambium, cortex, pith, leaf, and flower. In amost preferred embodiment, the location or tissue is a seed. In anotherpreferred embodiment, the nucleic acid encodes a polypeptide involved ina function such as, for example, but not limited to, carbon metabolism,photosynthesis, signal transduction, cell growth, reproduction, diseaseprocesses, gene regulation, and differentiation. In a more preferredembodiment, the nucleic acid encodes a polypeptide involved in abioticstress tolerance, enhanced yield, disease resistance, or nutritionalcontent.

In a preferred embodiment, the isolated nucleic acid comprising orconsisting of a nucleotide sequence capable of hybridizing to anucleotide sequence listed in SEQ ID No:1, or fragment, domain, orfeature thereof. In a preferred embodiment, hybridization allows thesequence to form a duplex atmedium or high stringency. Embodiments ofthe present invention also encompass a nucleotide sequence complementaryto a nucleotide sequence listed in SEQ ID No:1, or fragment, domain, orfeature thereof. Embodiments of the present invention further encompassa nucleotide sequence complementary to a nucleotide sequence that hassubstantial similarity or is capable of hybridizing to a nucleotidesequence listed in SEQ ID No:1, or fragment, domain, or feature thereof.

In a preferred embodiment, the nucleotide sequence having substantialsimilarity is an allelic variant of the nucleotide sequence listed inSEQ ID No:1, or fragment, domain, or feature thereof. In an alternateembodiment, the sequence having substantial similarity is a naturallyoccurring variant. In another alternate embodiment, the sequence havingsubstantial similarity is a polymorphic variant of the nucleotidesequence listed in SEQ ID No:1, or fragment, domain, or feature thereof.

In a preferred embodiment, the isolated nucleic acid contains aplurality of regions having the nucleotide sequence listed in SEQ IDNO:1, or exon, domain, or feature thereof.

In a preferred embodiment, the isolated nucleic acid contains apolypeptide-encoding sequence. In a more preferred embodiment, thepolypeptide-encoding sequence contains a 20 base pair nucleotide portionidentical in sequence to a consecutive 20 base pair nucleotide portionof a nucleic acid sequence listed in SEQ ID NO:1. In a more preferredembodiment, the polypeptide contains a polypeptide sequence listed inSEQ ID No:2, or a fragment thereof. In a more preferred embodiment, thepolypeptide is a plant polypeptide. In a more preferred embodiment, theplant is a dicot. In a more preferred embodiment, the plant is agymnosperm. In a more preferred embodiment, the plant is a monocot. In amore preferred embodiment, the monocot is a cereal. In a more preferredembodiment, the cereal may be, for example, maize, wheat, barley, oats,rye, millet, sorghum, triticale, secale, einkorn, spelt, emmer, teff,miloflax, gramma grass, Tripsacum, and teosinte. In a most preferredembodiment, the cereal is rice.

In one embodiment, the polypeptide is expressed throughout the plant. Ina more preferred embodiment, the polypeptide is expressed in a specificlocation or tissue of a plant. In a more preferred embodiment, thelocation or tissue may be, for example, epidermis, root, vasculartissue, meristem, cambium, cortex, pith, leaf, and flower. In a mostpreferred embodiment, the location or tissue is a seed.

In a preferred embodiment, the polypeptide is involved in a functionsuch as abiotic stress tolerance, enhanced yield, disease resistance ornutritional content.

In a preferred embodiment, the sequence of the isolated nucleic acidencodes a polypeptide useful for generating an antibody havingimmunoreactivity against a polypeptide encoded by a nucleotide sequencelisted in SEQ ID No:2, or fragment, domain, or feature thereof.

In a preferred embodiment, the sequence having substantial similaritycontains a deletion or insertion of at least one nucleotide. In a morepreferred embodiment, the deletion or insertion is of less than aboutthirty nucleotides. In a most preferred embodiment, the deletion orinsertion is of less than about five nucleotides.

In a preferred embodiment, the sequence of the isolated nucleic acidhaving substantial similarity comprises or consists of a substitution inat least one codon. In a preferred embodiment, the substitution isconservative.

Embodiments of the present invention also relate to the an isolatednucleic acid molecule comprising or consisting of a nucleotide sequence,its complement, or its reverse complement, encoding a polypeptideincluding:

-   -   (a) a polypeptide sequence listed in SEQ ID No:2, or a fragment,        domain, repeat, feature, or chimera thereof;    -   (b) a polypeptide sequence having substantial similarity to (a);    -   (c) a polypeptide sequence encoded by a nucleotide sequence        identical to or having substantial similarity to a nucleotide        sequence listed in SEQ ID No:1, or a fragment, domain, or        feature thereof, or a sequence complementary thereto;    -   (d) a polypeptide sequence encoded by a nucleotide sequence        capable of hybridizing under medium stringency conditions to a        nucleotide sequence listed in SEQ ID No:1, or to a sequence        complementary thereto; and    -   (e) a functional fragment of (a), (b), (c) or (d).

In another preferred embodiment, the polypeptide having substantialsimilarity is an allelic variant of a polypeptide sequence listed in SEQID NO:2, or a fragment, domain, repeat, feature, or chimeras thereof. Inanother preferred embodiment, the isolated nucleic acid includes aplurality of regions from the polypeptide sequence encoded by anucleotide sequence identical to or having substantial similarity to anucleotide sequence listed in SEQ ID NO:1, or fragment, domain, orfeature thereof, or a sequence complementary thereto.

In another preferred embodiment, the polypeptide is a polypeptidesequence listed in SEQ ID NO:2. In another preferred embodiment, thepolypeptide is a functional fragment or domain. In yet another preferredembodiment, the polypeptide is a chimera, where the chimera may includefunctional protein domains, including domains, repeats,post-translational modification sites, or other features. In a morepreferred embodiment, the polypeptide is a plant polypeptide. In a morepreferred embodiment, the plant is a dicot. In a more preferredembodiment, the plant is a gymnosperm. In a more preferred embodiment,the plant is a monocot. In a more preferred embodiment, the monocot is acereal. In a more preferred embodiment, the cereal may be, for example,maize, wheat, barley, oats, rye, millet, sorghum, triticale, secale,einkorn, spelt, emmer, teff, milo, flax, gramma grass, Tripsacum, andteosinte. In a most preferred embodiment, the cereal is rice.

In a preferred embodiment, the polypeptide is expressed in a specificlocation or tissue of a plant. In a more preferred embodiment, thelocation or tissue may be, for example, epidermis, root, vasculartissue, meristem, cambium, cortex, pith, leaf, and flower. In a morepreferred embodiment, the location or tissue is a seed.

In a preferred embodiment, the polypeptide is involved in a functionsuch as abiotic stress tolerance, disease resistance, enhanced yield ornutritional quality or composition.

In a preferred embodiment, the polypeptide sequence encoded by anucleotide sequence having substantial similarity to a nucleotidesequence listed in SEQ ID No:1 or a fragment, domain, or feature thereofor a sequence complementary thereto, includes a deletion or insertion ofat least one nucleotide. In a more preferred embodiment, the deletion orinsertion is of less than about thirty nucleotides. In a most preferredembodiment, the deletion or insertion is of less than about fivenucleotides.

In a preferred embodiment, the polypeptide sequence encoded by anucleotide sequence having substantial similarity to a nucleotidesequence listed in SEQ ID No:1, or fragment, domain, or feature thereofor a sequence complementary thereto, includes a substitution of at leastone codon. In a more preferred embodiment, the substitution isconservative.

In a preferred embodiment, the polypeptide sequences having substantialsimilarity to the polypeptide sequence listed in SEQ ID No:2, or afragment, domain, repeat, feature, or chimeras thereof includes adeletion or insertion of at least one amino acid.

In a preferred embodiment, the polypeptide sequences having substantialsimilarity to the polypeptide sequence listed in SEQ ID No:2, or afragment, domain, repeat, feature, or chimeras thereof includes asubstitution of at least one amino acid.

Embodiments of the present invention also relate to a shuffled nucleicacid containing a plurality of nucleotide sequence fragments, wherein atleast one of the fragments corresponds to a region of a nucleotidesequence listed in SEQ ID NO:1, and wherein at least two of theplurality of sequence fragments are in an order, from 5′ to 3′ which isnot an order in which the plurality of fragments naturally occur in anucleic acid. In a more preferred embodiment, all of the fragments in ashuffled nucleic acid containing a plurality of nucleotide sequencefragments are from a single gene. In a more preferred embodiment, theplurality of fragments originates from at least two different genes. Ina more preferred embodiment, the shuffled nucleic acid is operablylinked to a promoter sequence. Another more preferred embodiment is achimeric polynucleotide including a promoter sequence operably linked tothe shuffled nucleic acid. In a more preferred embodiment, the shufflednucleic acid is contained within a host cell.

Embodiments of the present invention also contemplate an expressioncassette including a promoter sequence operably linked to an isolatednucleic acid containing a nucleotide sequence including:

-   -   (a) a nucleotide sequence listed in SEQ ID NO:1, or fragment,        domain, or feature thereof;    -   (b) a nucleotide sequence having substantial similarity to (a);    -   (c) a nucleotide sequence capable of hybridizing to (a);    -   (d) a nucleotide sequence complementary to (a), (b) or (c); and    -   (e) a nucleotide sequence which is the reverse complement of        (a), (b) or (c).

Further encompassed within the invention, is a recombinant vectorcomprising an expression cassette according to embodiments of thepresent invention. Also encompassed are plant cells, which containexpression cassettes, according to the present disclosure, and plants,containing these plant cells. In a preferred embodiment, the plant is adicot. In another preferred embodiment, the plant is a gymnosperm. Inanother preferred embodiment, the plant is a monocot. In a morepreferred embodiment, the monocot is a cereal. In a more preferredembodiment, the cereal may be, for example, maize, wheat, barley, oats,rye, millet, sorghum, triticale, secale, einkorn, spelt, emmer, teff,milo, flax, gramma grass, Tripsacum and teosinte. In a most preferredembodiment, the cereal is rice.

In one embodiment, the expression cassette is expressed throughout theplant. In another embodiment, the expression cassette is expressed in aspecific location or tissue of a plant. In a preferred embodiment, thelocation or tissue may be, for example, epidermis, root, vasculartissue, meristem, cambium, cortex, pith, leaf, and flower. In a morepreferred embodiment, the location or tissue is a seed.

In one embodiment, the expression cassette is involved in a functionsuch as, for example, but not limited to, disease resistance, yield,abiotic stress resistance, nutritional quality, carbon metabolism,photosynthesis, signal transduction, cell growth, reproduction, diseaseprocesses, gene regulation, and differentiation. In a more preferredembodiment, the chimeric polypeptide is involved in a function such as,abiotic stress tolerance, enhanced yield, disease resistance ornutritional composition.

In one embodiment, the plant contains a modification to a phenotype ormeasurable characteristic of the plant, the modification beingattributable to theexpression cassette. In a preferred embodiment, themodification may be, for example, nutritional enhancement, increasednutrient uptake efficiency, enhanced production of endogenous compounds,and production of heterologous compounds. In another preferredembodiment, the modification includes having increased or decreasedresistance to an herbicide, a stress, or a pathogen. In anotherpreferred embodiment, the modification includes having enhanced ordiminished requirement for light, water, nitrogen, or trace elements. Inanother preferred embodiment, the modification includes being enrichedfor an essential amino acid as a proportion of a protein fraction of theplant. In a more preferred embodiment, the protein fraction may be, forexample, total seed protein, soluble protein, insoluble protein,water-extractable protein, and lipid-associated protein. In anotherpreferred embodiment, the modification includes overexpression,underexpression, antisense modulation, sense suppression, inducibleexpression, inducible repression, or inducible modulation of a gene.

Embodiments of the present invention also provide seed and isolatedproduct from plants which contain an expression cassette including apromoter sequence operably linked to an isolated nucleic acid containinga nucleotide sequence including:

-   -   (a) a nucleotide sequence listed in SEQ ID NO:1, or fragment,        domain, or feature thereof;    -   (b) a nucleotide sequence encoding a polypeptide of SEQ ID NO:2,        fragment, domain or feature thereof;    -   (c) a nucleotide sequence having substantial similarity to (a)        or (b);    -   (d) a nucleotide sequence capable of hybridizing to (a), (b) or        (c);    -   (e) a nucleotide sequence complementary to (a), (b), (c) or (d);        and    -   (f) a nucleotide sequence that is the reverse complement of (a),        (b), (c) or (d) according to the present disclosure.

Embodiments of the present invention also relate to isolated productsproduced by expression of an isolated nucleic acid containing anucleotide sequence including:

-   -   (a) a nucleotide sequence listed in SEQ ID NO:1, or fragment,        domain, or feature thereof;    -   (b) a nucleotide sequence encoding a polypeptide listed in SEQ        ID NO: 2, or fragment, domain or feature thereof;    -   (c) a nucleotide sequence having substantial similarity to (a)        or (b);    -   (d) a nucleotide sequence capable of hybridizing to (a) or (b);    -   (e) a nucleotide sequence complementary to (a), (b), (c) or (d);        and    -   (f) a nucleotide sequence that is the reverse complement of        (a), (b) (c) or (d) according to the present disclosure.

In a preferred embodiment, the product is produced in a plant. Inanother preferred embodiment, the product is produced in cell culture.In another preferred embodiment, the product is produced in a cell-freesystem. In another preferred embodiment, the product includes an enzyme,a nutritional protein, a structural protein, an amino acid, a lipid, afatty acid, a polysaccharide, a sugar, an alcohol, an alkaloid, acarotenoid, a propanoid, a steroid, a pigment, a vitamin and a planthormone.

In a preferred embodiment, the product is a polypeptide containing anamino acid sequence listed in SEQ ID NO:2. In a more preferredembodiment, the protein is an enzyme.

Embodiments of the present invention further relate to an isolatedpolynucleotide including a nucleotide sequence of at least 10 bases,which sequence is identical, complementary, or substantially similar toa region of any sequence of SEQ ID NO:1, and wherein the polynucleotideis adapted for any of numerous uses.

In a preferred embodiment, the polynucleotide is used as a chromosomalmarker. In another preferred embodiment, the polynucleotide is used as amarker for RFLP analysis. In another preferred embodiment, thepolynucleotide is used as a marker for quantitative trait linkedbreeding. In another preferred embodiment, the polynucleotide is used asa marker for marker-assisted breeding. In another preferred embodiment,the polynucleotide is used as a bait sequence in a two-hybrid system toidentify sequence-encoding polypeptides interacting with the polypeptideencoded by the bait sequence. In another preferred embodiment, thepolynucleotide is used as a diagnostic indicator for genotyping oridentifying an individual or population of individuals. In anotherpreferred embodiment, the polynucleotide is used for genetic analysis toidentify boundaries of genes or exons.

Embodiments of the present invention also relate to an expression vectorcomprising or consisting of a nucleic acid molecule including:

-   -   (a) a nucleic acid encoding a polypeptide as listed in SEQ ID        NO:2;    -   (b) a fragment, one or more domains, or featured regions listed        in SEQ ID NO:1; and    -   (c) a complete nucleic acid sequence listed in SEQ ID NO:1, or a        fragment thereof, in combination with a heterologous sequence.

In a preferred embodiment, the expression vector includes one or moreelements such as, for example, but not limited to, a promoter-enhancersequence, a selection marker sequence, an origin of replication, anepitope-tag encoding sequence, or an affinity purification-tag encodingsequence. In a more preferred embodiment, the promoter-enhancer sequencemay be, for example, the CaMV 35S promoter, the CaMV 19S promoter, thetobacco PR-1a promoter, ubiquitin and the phaseolin promoter. In anotherembodiment, the promoter is operable in plants, and more preferably, aconstitutive or inducible promoter. In another preferred embodiment, theselection marker sequence encodes an antibiotic resistance gene. Inanother preferred embodiment, the epitope-tag sequence encodes V5, thepeptide Phe-His-His-Thr-Thr, hemagglutinin, orglutathione-S-transferase. In another preferred embodiment the affinitypurification-tag sequence encodes a polyamino acid sequence or apolypeptide. In a more preferred embodiment, the polyamino acid sequenceis polyhistidine. In a more preferred embodiment, the polypeptide ischitin binding domain or glutathione-S-transferase. In a more preferredembodiment, the affinity purification-tag sequence comprises an inteinencoding sequence.

In a preferred embodiment, the expression vector is a eukaryoticexpression vector or a prokaryotic expression vector. In a morepreferred embodiment, the eukaryotic expression vector includes atissue-specific promoter. More preferably, the expression vector isoperable in plants.

Embodiments of the present invention also relate to a cell comprising orconsisting of a nucleic acid construct comprising an expression vectorand a nucleic acid including a nucleic acid encoding a polypeptide aslisted in SEQ ID NO:2, or a nucleic acid sequence listed in SEQ ID NO:1,or a segment thereof, in combination with a heterologous sequence.

In a preferred embodiment, the cell is a bacterial cell, a fungal cell,a plant cell, or an animal cell. In a more preferred embodiment, thepolypeptide is expressed in a specific location or tissue of a plant. Ina most preferred embodiment, the location or tissue may be, for example,epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf,and flower. In an alternate most preferred embodiment, the location ortissue is a seed. In a preferred embodiment, the polypeptide is involvedin a function such as, for example, carbon metabolism, photosynthesis,signal transduction, cell growth, reproduction, disease processes, generegulation, and differentiation. More preferably, the polypeptide isinvolved in a function such as, abiotic stress tolerance, enhancedyield, disease resistance or nutritional composition.

Embodiments of the present invention also relate to polypeptides encodedby the isolated nucleic acid molecules of the present disclosureincluding a polypeptide containing a polypeptide sequence encoded by anisolated nucleic acid containing a nucleotide sequence including:

-   -   (a) a nucleotide sequence listed in SEQ ID NO:1, or exon,        domain, or feature thereof;    -   (b) a nucleotide sequence having substantial similarity to (a);    -   (c) a nucleotide sequence capable of hybridizing to (a);    -   (d) a nucleotide sequence complementary to (a), (b) or (c); and    -   (e) a nucleotide sequence which is the reverse complement of        (a), (b) or (c);    -   (f) or a functional fragment thereof.

A polypeptide containing a polypeptide sequence encoded by an isolatednucleic acid containing a nucleotide sequence, its complement, or itsreverse complement, encoding a polypeptide including a polypeptidesequence including:

-   -   (a) a polypeptide sequence listed in SEQ ID NO:2, or a domain,        repeat, feature, or chimeras thereof;    -   (b) a polypeptide sequence having substantial similarity to (a);    -   (c) a polypeptide sequence encoded by a nucleotide sequence        identical to or having substantial similarity to a nucleotide        sequence listed in SEQ ID NO:1, or an exon, domain, or feature        thereof, or a sequence complementary thereto;    -   (d) a polypeptide sequence encoded by a nucleotide sequence        capable of hybridizing under medium stringency conditions to a        nucleotide sequence listed in SEQ ID NO:1, or to a sequence        complementary thereto; and    -   (e) a functional fragment of (a), (b), (c) or (d);    -   (f) or a functional fragment thereof.

Embodiments of the present invention contemplate a polypeptidecontaining a polypeptide sequence encoded by an isolated nucleic acidwhich includes a shuffled nucleic acid containing a plurality ofnucleotide sequence fragments, wherein at least one of the fragmentscorresponds to a region of a nucleotide sequence listed in SEQ ID NO:1,and wherein at least two of the plurality of sequence fragments are inan order, from 5′ to 3′ which is not an order in which the plurality offragments naturally occur in a nucleic acid, or functional fragmentthereof.

Embodiments of the present invention contemplate a polypeptidecontaining a polypeptide sequence encoded by an isolated polynucleotidecontaining a nucleotide sequence of at least 10 bases, which sequence isidentical, complementary, or substantially similar to a region of any ofsequences of SEQ ID NO:1, and wherein the polynucleotide is adapted fora use including:

-   -   (a) use as a chromosomal marker to identify the location of the        corresponding or complementary polynucleotide on a native or        artificial chromosome;    -   (b) use as a marker for RFLP analysis;    -   (c) use as a marker for quantitative trait linked breeding;    -   (d) use as a marker for marker-assisted breeding;    -   (e) use as a bait sequence in a two-hybrid system to identify        sequence encoding polypeptides interacting with the polypeptide        encoded by the bait sequence;    -   (f) use as a diagnostic indicator for genotyping or identifying        an individual or population of individuals; and    -   (g) use for genetic analysis to identify boundaries of genes or        exons;    -   (h) or functional fragment thereof.

Embodiments of the present invention also contemplate an isolatedpolypeptide containing a polypeptide sequence including:

-   -   (a) a polypeptide sequence listed in SEQ ID NO:2, or exon,        domain, or feature thereof;    -   (b) a polypeptide sequence having substantial similarity to (a);    -   (c) a polypeptide sequence encoded by a nucleotide sequence        identical to or having substantial similarity to a nucleotide        sequence listed in SEQ ID NO:1, or an exon, domain, or feature        thereof, or a sequence complementary thereto;    -   (d) a polypeptide sequence encoded by a nucleotide sequence        capable of hybridizing under medium stringency conditions to a        nucleotide sequence listed in SEQ ID NO:1, or to a sequence        complementary thereto; and    -   (e) a functional fragment of (a), (b), (c) or (d).

In a preferred embodiment, the substantial similarity is at least about65% identity. In a more preferred embodiment, the substantial similarityis at least about 80% identity. In a most preferred embodiment, thesubstantial similarity is at least about 95% identity. In a preferredembodiment, the substantial similarity is at least three percent greaterthan the percent identity to the closest homologous sequence listed inany of the Tables.

In a preferred embodiment, the sequence having substantial similarity isfrom a plant. In a more preferred embodiment, the plant is a dicot. In amore preferred embodiment, the plant is a gymnosperm. In a morepreferred embodiment, the plant is a monocot. In a more preferredembodiment, the monocot is a cereal. In a more preferred embodiment, thecereal may be, for example, maize, wheat, barley, oats, rye, millet,sorghum, triticale, secale, einkorn, spelt, emmer, teff, milo, flax,gramma grass, Tripsacum and teosinte. In a most preferred embodiment,the cereal is rice.

In a preferred embodiment, the polypeptide is expressed in a specificlocation or tissue of a plant. In a more preferred embodiment, thelocation or tissue may be, for example, epidermis, root, vasculartissue, meristem, cambium, cortex, pith, leaf, and flower. In a morepreferred embodiment, the location or tissue is a seed. In a preferredembodiment, the polypeptide is involved in a function such as, forexample, carbon metabolism, photosynthesis, signal transduction, cellgrowth, reproduction, disease processes, gene regulation, anddifferentiation.

In a preferred embodiment, hybridization of a polypeptide sequenceencoded by a nucleotide sequence identical to or having substantialsimilarity to a nucleotide sequence listed in SEQ ID NO:1, or an exon,domain, or feature thereof, or a sequence complementary thereto, or apolypeptide sequence encoded by a nucleotide sequence capable ofhybridizing under medium stringency conditions to a nucleotide sequencelisted in SEQ ID NO:1, or to a sequence complementary thereto, allowsthe sequence to form a duplex atmedium or high stringency.

In a preferred embodiment, a polypeptide having substantial similarityto a polypeptide sequence listed in SEQ ID NO:2, or exon, domain, orfeature thereof, is an allelic variant of the polypeptide sequencelisted in SEQ ID NO:2. In another preferred embodiment, a polypeptidehaving substantial similarity to a polypeptide sequence listed in SEQ IDNO:2, or exon, domain, or feature thereof, is a naturally occurringvariant of the polypeptide sequence listed in SEQ ID NO:2. In anotherpreferred embodiment, a polypeptide having substantial similarity to apolypeptide sequence listed in SEQ ID NO:2, or exon, domain, or featurethereof, is a polymorphic variant of the polypeptide sequence listed inSEQ ID NO:2.

In an alternate preferred embodiment, the sequence having substantialsimilarity contains a deletion or insertion of at least one amino acid.In a more preferred embodiment, the deletion or insertion is of lessthan about ten amino acids. In a most preferred embodiment, the deletionor insertion is of less than about three amino acids.

In a preferred embodiment, the sequence having substantial similarityencodes a substitution in at least one amino acid.

Also contemplated is a method of producing a plant having enhancedtolerance to biotic and/or abiotic stress comprising the steps of:

-   -   (1) providing a nucleic acid which is an isolated nucleic acid        containing a nucleotide sequence including:    -   (a) a nucleotide sequence listed in SEQ ID NO:1, or exon,        domain, or feature thereof;    -   (b) a nucleotide sequence having substantial similarity to (a);    -   (c) a nucleotide sequence capable of hybridizing to (a);    -   (d) a nucleotide sequence complementary to (a), (b) or (c); and    -   (e) a nucleotide sequence which is the reverse complement of        (a), (b) or (c);        and (2) introducing the nucleic acid into the plant, wherein the        nucleic acid is expressible in the plant in an amount effective        to enhance the tolerance to abiotic stress.

Also encompassed within the presently disclosed invention is a method ofproducing a recombinant protein, comprising the steps of:

(a) growing recombinant cells comprising a nucleic acid construct Undersuitable growth conditions, the construct comprising an expressionvector and a nucleic acid including: a nucleic acid encoding a proteinas listed in SEQ ID NO:2, or a nucleic acid sequence listed in SEQ IDNOS:1, or segments thereof; and

(b) isolating from the recombinant cells the recombinant proteinexpressed thereby.

Embodiments of the present invention provide a method of producing arecombinant protein in which the expression vector includes one or moreelements including a promoter-enhancer sequence, a selection markersequence, an origin of replication, an epitope-tag encoding sequence,and an affinity purification-tag encoding sequence. In one preferredembodiment, the nucleic acid construct includes an epitope-tag encodingsequence and the isolating step includes use of an antibody specific forthe epitope-tag. In another preferred embodiment, the nucleic acidconstruct contains a polyamino acid encoding sequence and the isolatingstep includes use of a resin comprising a polyamino acid bindingsubstance, preferably where the polyamino acid is polyhistidine and thepolyamino binding resin is nickel-charged agarose resin. In yet anotherpreferred embodiment, the nucleic acid construct contains a polypeptideencoding sequence and the isolating step includes the use of a resincontaining a polypeptide binding substance, preferably where thepolypeptide is a chitin binding domain and the resin containschitin-sepharose.

Embodiments of the present invention also relate to a plant modified bya method that includes introducing into a plant a nucleic acid where thenucleic acid is expressible in the plant in an amount effective toeffect the modification. The modification can be, for example,nutritional enhancement, increased nutrient uptake efficiency, enhancedproduction of endogenous compounds, and production of heterologouscompounds. In one embodiment, the modified plant has increased ordecreased resistance to an herbicide, a stress, or a pathogen. Inanother embodiment, the modified plant has enhanced or diminishedrequirement for light, water, nitrogen, or trace elements. In yetanother embodiment, the modified plant is enriched for an essentialamino acid as a proportion of a protein fraction of the plant. Theprotein fraction may be, for example, total seed protein, solubleprotein, insoluble protein, water-extractable protein, andlipid-associated protein. The modification may include overexpression,underexpression, antisense modulation, sense suppression, inducibleexpression, inducible repression, or inducible modulation of a gene.

The invention further relates to a seed from a modified plant or anisolated product of a modified plant, where the product may be anenzyme, a nutritional protein, a structural protein, an amino acid, alipid, a fatty acid, a polysaccharide, a sugar, an alcohol, an alkaloid,a carotenoid, a propanoid, a steroid, a pigment, a vitamin and a planthormone.

The invention further relates to an isolated nucleic acid moleculecapable of driving biotic and/or abiotic stress responsive expression ofan associated nucleotide sequence in particular, wherein said isolatednucleic acid molecule

-   -   a) is a component of SEQ ID NO:4;    -   b) is depicted in SEQ ID NO:4;    -   c) comprises the nucleotide sequence depicted in SEQ ID NO:4;    -   d) hybridizes under stringent conditions to SEQ ID NO:4 or SEQ        ID NO:3, wherein the nucleic acid molecule is capable of driving        stress responsive expression of an associated nucleotide        sequence; or    -   e) comprises a consecutive stretch of at least 50 nt, preferably        of about 500 bases, particularly of between about 1000 bases and        about 1500 bases, and more particularly of about 1551 bases SEQ        ID NO:4, wherein said isolated nucleic acid molecule is capable        of driving abiotic stress responsive expression of an associated        nucleotide sequence, in particular, wherein said consecutive        stretch of at least 50 nt has at least 70%, preferably 80%, more        preferably 90% and most preferably 95% sequence identity        sequence identity with a consecutive stretch of corresponding        length of SEQ ID NO:4.        The invention further provides for an isolated nucleic acid        molecule comprising the sequence of SEQ ID NO:3 which encodes        the genomic clone of the Arabidopsis thaliana BOS1 gene.

For purposes of summarizing the invention and the advantages achievedover the prior art, certain objects and advantages of the invention havebeen described above. Of course, it is to be understood that notnecessarily all such objects or advantages may be achieved in accordancewith any particular embodiment of the invention. Thus, for example,those skilled in the art will recognize that the invention may beembodied or carried out in a manner that achieves or optimizes oneadvantage or group of advantages as taught herein without necessarilyachieving other objects or advantages as may be taught or suggestedherein.

Further aspects, features and advantages of this invention will becomeapparent from the detailed description of the preferred embodiments thatfollow.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LIST

-   SEQ ID NO:1 is the cDNA nucleotide sequence of the BOS1 gene from    Arabidopsis thaliana.-   SEQ ID NO:2 is the deduced amino acid sequence of the BOS1 protein    encoded by SEQ ID NO:1.-   SEQ ID NO:3 is the genomic nucleotide sequence of the BOS1 gene from    Arabidopsis thaliana.-   SEQ ID NO:4 is the nucleotide sequence of the promoter of the BOS1    gene from Arabidopsis thaliana.-   SEQ ID NO:5 is the 3′ RACE primer 1.-   SEQ ID NO:6 is the 3′RACE primer 2.-   SEQ ID NO:7 is the 5′ RACE primer 1.-   SEQ ID NO:8 is the 5′ RACE primer 2.-   SEQ ID NO:9 is the BOS1 cDNA forward primer.-   SEQ ID NO:10 is the BOS1 cDNA reverse primer.-   SEQ ID NO:11 is the BOS1 genomic forward primer.-   SEQ ID NO:12 is the BOS1 genomic reverse primer.-   SEQ ID NO:13 is the BOS1 promoter forward primer.-   SEQ ID NO:14 is the BOS1 promoter reverse primer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a botrytis susceptible (bos1) mutant from a T-DNAmutagenized population based on its increased susceptibility to Botrytiscinerea compared to wild type parental ecotype Col-0 plants.

FIG. 2 shows the bos1 mutant (7675–30) to have increased susceptiblityto another necrotrophic pathogen, Alternaria brassicicola compared towild type (7675–29).

FIG. 3 shows the bos1 mutant to have increased susceptibility to abacterial pathogen, Pseudomonas syringae compared to wild type.

FIG. 4 is a graph showing that the survival of bos1 plants wassignificantly reduced following exposure to salt stress. When grown inthe presence of 100 mM NaCl, only 18.75% of bos1 plants survivedcompared to 93.75% of wild type plants.

FIG. 5 shows that the bos1 plants are sensitive to drought stresscompared to wild type plants.

FIG. 6 shows that the bos1 mutant was affected in the normal expressionof the gene encoding for a MYB transcription factor (referred to asAtMYB108 in the public data base, referred to herein as BOS1) due to aT-DNA insertion.

FIGS. 7A & B show that constitutive expression of BOS1 (AtMYB108) underthe regulation of the Arabidopsis UBQ3 promoter restores the Botrytissusceptibility phenotype of the mutant to the wild type level ofresistance.

FIG. 8 shows that the BOS1 gene was induced following Botrytisinfection.

DEFINITIONS

For clarity, certain terms used in the specification are defined andpresented as follows:

“Associated with/operatively linked” refer to two nucleic acid sequencesthat are related physically or functionally. For example, a promoter orregulatory DNA sequence is said to be “associated with” a DNA sequencethat codes for an RNA or a protein if the two sequences are operativelylinked, or situated such that the regulator DNA sequence will affect theexpression level of the coding or structural DNA sequence.

A “chimeric construct” is a recombinant nucleic acid sequence in which apromoter or regulatory nucleic acid sequence is operatively linked to,or associated with, a nucleic acid sequence that codes for an mRNA orwhich is expressed as a protein, such that the regulatory nucleic acidsequence is able to regulate transcription or expression of theassociated nucleic acid sequence. The regulatory nucleic acid sequenceof the chimeric construct is not normally operatively linked to theassociated nucleic acid sequence as found in nature.

A “co-factor” is a natural reactant, such as an organic molecule or ametal ion, required in an enzyme-catalyzed reaction. A co-factor is e.g.NAD(P), riboflavin (including FAD and FMN), folate, molybdopterin,thiamin, biotin, lipoic acid, pantothenic acid and coenzyme A,S-adenosylmethionine, pyridoxal phosphate, ubiquinone, menaquinone.Optionally, a co-factor can be regenerated and reused.

A “coding sequence” is a nucleic acid sequence that is transcribed intoRNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA.Preferably the RNA is then translated in an organism to produce aprotein.

Complementary: “complementary” refers to two nucleotide sequences thatcomprise antiparallel nucleotide sequences capable of pairing with oneanother upon formation of hydrogen bonds between the complementary baseresidues in the antiparallel nucleotide sequences.

Enzyme activity: means herein the ability of an enzyme to catalyze theconversion of a substrate into a product. A substrate for the enzymecomprises the natural substrate of the enzyme but also comprisesanalogues of the natural substrate, which can also be converted, by theenzyme into a product or into an analogue of a product. The activity ofthe enzyme is measured for example by determining the amount of productin the reaction after a certain period of time, or by determining theamount of substrate remaining in the reaction mixture after a certainperiod of time. The activity of the enzyme is also measured bydetermining the amount of an unused co-factor of the reaction remainingin the reaction mixture after a certain period of time or by determiningthe amount of used co-factor in the reaction mixture after a certainperiod of time. The activity of the enzyme is also measured bydetermining the amount of a donor of free energy or energy-rich molecule(e.g. ATP, phosphoenolpyruvate, acetyl phosphate or phosphocreatine)remaining in the reaction mixture after a certain period of time or bydetermining the amount of a used donor of free energy or energy-richmolecule (e.g. ADP, pyruvate, acetate or creatine) in the reactionmixture after a certain period of time.

Expression Cassette: “Expression cassette” as used herein means anucleic acid molecule capable of directing expression of a particularnucleotide sequence in an appropriate host cell, comprising a promoteroperatively linked to the nucleotide sequence of interest which isoperatively linked to termination signals. It also typically comprisessequences required for proper translation of the nucleotide sequence.The coding region usually codes for a protein of interest but may alsocode for a functional RNA of interest, for example antisense RNA or anontranslated RNA, in the sense or antisense direction. The expressioncassette comprising the nucleotide sequence of interest may be chimeric,meaning that at least one of its components is heterologous with respectto at least one of its other components. The expression cassette mayalso be one that is naturally occurring but has been obtained in arecombinant form useful for heterologous expression. Typically, however,the expression cassette is heterologous with respect to the host, i.e.,the particular DNA sequence of the expression cassette does not occurnaturally in the host cell and must have been introduced into the hostcell or an ancestor of the host cell by a transformation event. Theexpression of the nucleotide sequence in the expression cassette may beunder the control of a constitutive promoter or of an inducible promoterthat initiates transcription only when the host cell is exposed to someparticular external stimulus. In the case of a multicellular organism,such as a plant, the promoter can also be specific to a particulartissue or organ or stage of development.

Gene: the term “gene” is used broadly to refer to any segment of DNAassociated with a biological function. Thus, genes include codingsequences and/or the regulatory sequences required for their expression.Genes also include nonexpressed DNA segments that, for example, formrecognition sequences for other proteins. Genes can be obtained from avariety of sources, including cloning from a source of interest orsynthesizing from known or predicted sequence information, and mayinclude sequences designed to have desired parameters.

Heterologous/exogenous: The terms “heterologous” and “exogenous” whenused herein to refer to a nucleic acid sequence (e.g. a DNA sequence) ora gene, refer to a sequence that originates from a source foreign to theparticular host cell or, if from the same source, is modified from itsoriginal form. Thus, a heterologous gene in a host cell includes a genethat is endogenous to the particular host cell but has been modifiedthrough, for example, the use of DNA shuffling. The terms also includenon-naturally occurring multiple copies of a naturally occurring DNAsequence. Thus, the terms refer to a DNA segment that is foreign orheterologous to the cell, or homologous to the cell but in a positionwithin the host cell nucleic acid in which the element is not ordinarilyfound. Exogenous DNA segments are expressed to yield exogenouspolypeptides.

A “homologous” nucleic acid (e.g. DNA) sequence is a nucleic acid (e.g.DNA) sequence naturally associated with a host cell into which it isintroduced.

Hybridization: The phrase “hybridizing specifically to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent conditions when that sequence ispresent in a complex mixture (e.g., total cellular) DNA or RNA. “Bind(s)substantially” refers to complementary hybridization between a probenucleic acid and a target nucleic acid and embraces minor mismatchesthat can be accommodated by reducing the stringency of the hybridizationmedia to achieve the desired detection of the target nucleic acidsequence.

Inhibitor: a chemical substance that inactivates the enzymatic activityof a protein such as a biosynthetic enzyme, receptor, signaltransduction protein, structural gene product, or transport protein. Theterm “herbicide” (or “herbicidal compound”) is used herein to define aninhibitor applied to a plant at any stage of development, whereby theherbicide inhibits the growth of the plant or kills the plant.

Interaction: quality or state of mutual action such that theeffectiveness or toxicity of one protein or compound on another proteinis inhibitory (antagonists) or enhancing (agonists).

A nucleic acid sequence is “isocoding with” a reference nucleic acidsequence when the nucleic acid sequence encodes a polypeptide having thesame amino acid sequence as the polypeptide encoded by the referencenucleic acid sequence.

Isogenic: plants that are genetically identical, except that they maydiffer by the presence or absence of a heterologous DNA sequence.

Isolated: in the context of the present invention, an isolated DNAmolecule or an isolated enzyme is a DNA molecule or enzyme that, by thehand of man, exists apart from its native environment and is thereforenot a product of nature. An isolated DNA molecule or enzyme may exist ina purified form or may exist in a non-native environment such as, forexample, in a transgenic host cell.

Mature protein: protein from which the transit peptide, signal peptide,and/or propeptide portions have been removed.

Minimal Promoter: the smallest piece of a promoter, such as a TATAelement, that can support any transcription. A minimal promotertypically has greatly reduced promoter activity in the absence ofupstream activation. In the presence of a suitable transcription factor,the minimal promoter functions to permit transcription.

Modified Enzyme Activity: enzyme activity different from that whichnaturally occurs in a plant (i.e. enzyme activity that occurs naturallyin the absence of direct or indirect manipulation of such activity byman), which is tolerant to inhibitors that inhibit the naturallyoccurring enzyme activity.

Native: refers to a gene that is present in the genome of anuntransformed plant cell.

Naturally occurring: the term “naturally occurring” is used to describean object that can be found in nature as distinct from beingartificially produced by man. For example, a protein or nucleotidesequence present in an organism (including a virus), which can beisolated from a source in nature and which has not been intentionallymodified by man in the laboratory, is naturally occurring.

Nucleic acid: the term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.degenerate codon substitutions) and complementary sequences and as wellas the sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605–2608(1985); Rossolini et al., Mol. Cell. Probes 8: 91–98 (1994)). The terms“nucleic acid” or “nucleic acid sequence” may also be usedinterchangeably with gene, cDNA, and mRNA encoded by a gene.

“ORF” means open reading frame.

Percent identity: the phrases “percent identical” or “percentidentical,” in the context of two nucleic acid or protein sequences,refers to two or more sequences or subsequences that have for example60%, preferably 70%, more preferably 80%, still more preferably 90%,even more preferably 95%, and most preferably at least 99% nucleotide oramino acid residue identity, when compared and aligned for maximumcorrespondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. Preferably, the percentidentity exists over a region of the sequences that is at least about 50residues in length, more preferably over a region of at least about 100residues, and most preferably the percent identity exists over at leastabout 150 residues. In an especially preferred embodiment, the percentidentity exists over the entire length of the coding regions.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch,J. Mol. Biol. 48: 443 (1970), by the search for similarity method ofPearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection (seegenerally, Ausubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215: 403–410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information at the World Wide Web sitencbi.nlm.nih.gov. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., 1990). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always>0)and N (penalty score for mismatching residues; always>0). For amino acidsequences, a scoring matrix is used to calculate the cumulative score.Extension of the word hits in each direction are halted when thecumulative alignment score falls off by the quantity X from its maximumachieved value, the cumulative score goes to zero or below due to theaccumulation of one or more negative-scoring residue alignments, or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad.Sci. USA 89: 10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90: 5873–5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a test nucleicacid sequence is considered similar to a reference sequence if thesmallest sum probability in a comparison of the test nucleic acidsequence to the reference nucleic acid sequence is less than about 0.1,more preferably less than about 0.01, and most preferably less thanabout 0.001.

Pre-protein: protein that is normally targeted to a cellular organelle,such as a chloroplast, and still comprises its native transit peptide.

Promoter: refers to a DNA sequence that initiates transcription of anassociated DNA sequence. The promoter region may also include elementsthat act as regulators of gene expression such as activators, enhancers,and/or repressors and may include all or part of the 5′ non-translatedregion.

Purified: the term “purified,” when applied to a nucleic acid orprotein, denotes that the nucleic acid or protein is essentially free ofother cellular components with which it is associated in the naturalstate. It is preferably in a homogeneous state although it can be ineither a dry or aqueous solution. Purity and homogeneity are typicallydetermined using analytical chemistry techniques such as polyacrylamidegel electrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified. The term “purified” denotes that a nucleic acidor protein gives rise to essentially one band in an electrophoretic gel.Particularly, it means that the nucleic acid or protein is at leastabout 50% pure, more preferably at least about 85% pure, and mostpreferably at least about 99% pure.

Two nucleic acids are “recombined” when sequences from each of the twonucleic acids are combined in a progeny nucleic acid. Two sequences are“directly” recombined when both of the nucleic acids are substrates forrecombination. Two sequences are “indirectly recombined” when thesequences are recombined using an intermediate such as a cross-overoligonucleotide. For indirect recombination, no more than one of thesequences is an actual substrate for recombination, and in some cases,neither sequence is a substrate for recombination.

“Regulatory elements” refer to sequences involved in controlling theexpression of a nucleotide sequence. Regulatory elements comprise apromoter operatively linked to the nucleotide sequence of interest andtermination signals. They also typically encompass sequences requiredfor proper translation of the nucleotide sequence.

Significant Increase: an increase in enzymatic activity that is largerthan the margin of error inherent in the measurement technique,preferably an increase by about 2-fold or greater of the activity of thewild-type enzyme in the presence of the inhibitor, more preferably anincrease by about 5-fold or greater, and most preferably an increase byabout 10-fold or greater.

Significantly less: means that the amount of a product of an enzymaticreaction is reduced by more than the margin of error inherent in themeasurement technique, preferably a decrease by about 2-fold or greaterof the activity of the wild-type enzyme in the absence of the inhibitor,more preferably an decrease by about 5-fold or greater, and mostpreferably an decrease by about 10-fold or greater.

Specific Binding/Immunological Cross-Reactivity: An indication that twonucleic acid sequences or proteins are substantially identical is thatthe protein encoded by the first nucleic acid is immunologically crossreactive with, or specifically binds to, the protein encoded by thesecond nucleic acid. Thus, a protein is typically substantiallyidentical to a second protein, for example, where the two proteinsdiffer only by conservative substitutions. The phrase “specifically (orselectively) binds to an antibody,” or “specifically (or selectively)immunoreactive with,” when referring to a protein or peptide, refers toa binding reaction which is determinative of the presence of the proteinin the presence of a heterogeneous population of proteins and otherbiologics. Thus, under designated immunoassay conditions, the specifiedantibodies bind to a particular protein and do not bind in a significantamount to other proteins present in the sample. Specific binding to anantibody under such conditions may require an antibody that is selectedfor its specificity for a particular protein. For example, antibodiesraised to the protein with the amino acid sequence encoded by any of thenucleic acid sequences of the invention can be selected to obtainantibodies specifically immunoreactive with that protein and not withother proteins except for polymorphic variants. A variety of immunoassayformats may be used to select antibodies specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassays,Western blots, or immunohistochemistry are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. SeeHarlow and Lane (1988) Antibodies, A Laboratory Manual, Cold SpringHarbor Publications, New York “Harlow and Lane”), for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity. Typically a specific or selective reactionwill be at least twice background signal or noise and more typicallymore than 10 to 100 times background.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridizations are sequence dependent, andare different under different environmental parameters. Longer sequenceshybridize specifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology-Hybridization withNucleic Acid Probes part I chapter 2 “Overview of principles ofhybridization and the strategy of nucleic acid probe assays” Elsevier,N.Y. Generally, highly stringent hybridization and wash conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence at a defined ionic strength and pH. Typically,under “stringent conditions” a probe will hybridize to its targetsubsequence, but to no other sequences.

The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Very stringent conditions are selected to be equal to the T_(m)for a particular probe. An example of stringent hybridization conditionsfor hybridization of complementary nucleic acids which have more than100 complementary residues on a filter in a Southern or northern blot is50% formamide with 1 mg of heparin at 42° C., with the hybridizationbeing carried out overnight. An example of highly stringent washconditions is 0.1 5 M NaCl at 72° C. for about 15 minutes. An example ofstringent wash conditions is a 0.2×SSC wash at 65° C. for 15 minutes(see, Sambrook, infra, for a description of SSC buffer). Often, a highstringency wash is preceded by a low stringency wash to removebackground probe signal. An example medium stringency wash for a duplexof, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for 15 minutes.An example low stringency wash for a duplex of, e.g., more than 100nucleotides, is 4–6×SSC at 40° C. for 15 minutes. For short probes(e.g., about 10 to 50 nucleotides), stringent conditions typicallyinvolve salt concentrations of less than about 1.0 M Na ion, typicallyabout 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to8.3, and the temperature is typically at least about 30° C. Stringentconditions can also be achieved with the addition of destabilizingagents such as formamide. In general, a signal to noise ratio of 2× (orhigher) than that observed for an unrelated probe in the particularhybridization assay indicates detection of a specific hybridization.Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the proteins that theyencode are substantially identical. This occurs, e.g., when a copy of anucleic acid is created using the maximum codon degeneracy permitted bythe genetic code.

The following are examples of sets of hybridization/wash conditions thatmay be used to clone nucleotide sequences that are homologues ofreference nucleotide sequences of the present invention: a referencenucleotide sequence preferably hybridizes to the reference nucleotidesequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at50° C. with washing in 2×SSC, 0.1% SDS at 50° C., more desirably in 7%sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. withwashing in 1×SSC, 0.1% SDS at 50° C., more desirably still in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodium dodecyl sulfate(SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1%SDS at 50° C., more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 MNaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 65° C.

A “subsequence” refers to a sequence of nucleic acids or amino acidsthat comprise a part of a longer sequence of nucleic acids or aminoacids (e.g., protein) respectively.

Substrate: a substrate is the molecule that an enzyme naturallyrecognizes and converts to a product in the biochemical pathway in whichthe enzyme naturally carries out its function, or is a modified versionof the molecule, which is also recognized by the enzyme and is convertedby the enzyme to a product in an enzymatic reaction similar to thenaturally-occurring reaction.

Transformation: a process for introducing heterologous DNA into a plantcell, plant tissue, or plant. Transformed plant cells, plant tissue, orplants are understood to encompass not only the end product of atransformation process, but also transgenic progeny thereof.

“Transformed,” “transgenic,” and “recombinant” refer to a host organismsuch as a bacterium or a plant into which a heterologous nucleic acidmolecule has been introduced. The nucleic acid molecule can be stablyintegrated into the genome of the host or the nucleic acid molecule canalso be present as an extrachromosomal molecule. Such anextrachromosomal molecule can be auto-replicating. Transformed cells,tissues, or plants are understood to encompass not only the end productof a transformation process, but also transgenic progeny thereof. A“non-transformed,” “non-transgenic,” or “non-recombinant” host refers toa wild-type organism, e.g., a bacterium or plant, which does not containthe heterologous nucleic acid molecule.

Viability: “viability” as used herein refers to a fitness parameter of aplant. Plants are assayed for their homozygous performance of plantdevelopment, indicating which proteins are essential for plant growth.

DETAILED DESCRIPTION OF THE INVENTION

I. General Description of Trait Functional Genomics Project

The goal of functional genomics is to identify genes controllingexpression of organismal phenotypes, and employs a variety ofmethodologies, including but not limited to bioinformatics, geneexpression studies, gene and gene product interactions, genetics,biochemistry and molecular genetics. For example, bioinformatics canassign function to a given gene by identifying genes in heterologousorganisms with a high degree of similarity (homology) at the amino acidor nucleotide level. Expression of a gene at the mRNA or protein levelscan assign function by linking expression of a gene to an environmentalresponse, a developmental process or a genetic (mutational) or moleculargenetic (gene overexpression or underexpression) perturbation.Expression of a gene at the mRNA level can be ascertained either alone(Northern analysis) or in concert with other genes (microarrayanalysis), whereas expression of a gene at the protein level can beascertained either alone (native or denatured protein gel or immunoblotanalysis) or in concert with other genes (proteomic analysis). Knowledgeof protein/protein and protein/DNA interactions can assign function byidentifying proteins and nucleic acid sequences acting together in thesame biological process. Genetics can assign function to a gene bydemonstrating that DNA lesions (mutations) in the gene have aquantifiable effect on the organism, including but not limited to: itsdevelopment; hormone biosynthesis and response; growth and growth habit(plant architecture); mRNA expression profiles; protein expressionprofiles; ability to resist diseases; tolerance of abiotic stresses;ability to acquire nutrients; photosynthetic efficiency; altered primaryand secondary metabolism; and the composition of various plant organs.Biochemistry can assign function by demonstrating that the proteinencoded by the gene, typically when expressed in a heterologousorganism, possesses a certain enzymatic activity, alone or incombination with other proteins. Molecular genetics can assign functionby overexpressing or underexpressing the gene in the native plant or inheterologous organisms, and observing quantifiable effects as describedin functional assignment by genetics above. In functional genomics, anyor all of these approaches are utilized, often in concert, to assigngenes to functions across any of a number of organismal phenotypes.

It is recognized by those skilled in the art that these differentmethodologies can each provide data as evidence for the function of aparticular gene, and that such evidence is stronger with increasingamounts of data used for functional assignment: preferably from a singlemethodology, more preferably from two methodologies, and even morepreferably from more than two methodologies. In addition, those skilledin the art are aware that different methodologies can differ in thestrength of the evidence for the assignment of gene function. Typically,but not always, a datum of biochemical, genetic and molecular geneticevidence is considered stronger than a datum of bioinformatic or geneexpression evidence. Finally, those skilled in the art recognize that,for different genes, a single datum from a single methodology can differin terms of the strength of the evidence provided by each distinct datumfor the assignment of the function of these different genes.

The objective of crop trait functional genomics is to identify croptrait genes, i.e. genes capable of conferring useful agronomic traits incrop plants. Such agronomic traits include, but are not limited to:enhanced yield, whether in quantity or quality; enhanced nutrientacquisition and enhanced metabolic efficiency; enhanced or alterednutrient composition of plant tissues used for food, feed, fiber orprocessing; enhanced utility for agricultural or industrial processing;enhanced resistance to plant diseases; enhanced tolerance of adverseenvironmental conditions (abiotic stresses) including but not limited todrought, excessive cold, excessive heat, or excessive soil salinity orextreme acidity or alkalinity; and alterations in plant architecture ordevelopment, including changes in developmental timing. The deploymentof such identified trait genes by either transgenic or non-transgenicmeans could materially improve crop plants for the benefit ofagriculture.

II. Identifying, Cloning and Sequencing cDNAs

The cloning and sequencing of the cDNAs of the present invention aredescribed in the Examples below.

The isolated nucleic acids and proteins of the present invention areusable over a range of plants, monocots and dicots, in particularmonocots such as rice, wheat, barley and maize. In a more preferredembodiment, the monocot is a cereal. In a more preferred embodiment, thecereal may be, for example, maize, wheat, barley, oats, rye, millet,sorghum, triticale, secale, einkorn, spelt, emmer, teff, milo, flax,gramma grass, Tripsacum sp., or teosinte. In a most preferredembodiment, the cereal is rice. Other plants genera include, but are notlimited to, Cucurbita, Rosa, Vitis, Juglans, Gragaria, Lotus, Medicago,Onobrychis, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus,Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura,Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis,Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus,Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum,Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum,Phaseolus, Lolium, Oryza, Avena, Hordeum, Secale, Allium, and Triticum.

The present invention also provides a method of genotyping a plant orplant part comprising a nucleic acid molecule of the present invention.Optionally, the plant is a monocot such as, but not limited rice orwheat. Genotyping provides a means of distinguishing homologs of achromosome pari and can be used to differentiate segregants in a plantpopulation. Molecular marker methods can be used in phylogeneticstudies, characterizing genetic relationships among crop varieties,identifying crosses or somatic hybrids, localizing chromosomeal segmentsaffecting mongenic traits, map based cloning, and the study ofquantitative inheritance (see Plant Molecular Biology: A LaboratoryManual, Chapter 7, Clark ed., Springer-Verlag, Berlin 1997; Paterson, A.H., “The DNA Revolution”, chapter 2 in Genome Mapping in Plants,Paterson, A. H. ed., Academic Press/R. G. Lands Co., Austin, Tex. 1996).

The method of genotyping may employ any number of molecular markeranalytical techniques such as, but not limited to, restriction lengthpolymorphisms (RFLPs). As is well known in the art, RFLPs are producedby differences in the DNA restriction fragment lengths resulting fromnucleotide differences between alleles of the same gene. Thus, thepresent invention provides a method of following segregation of a geneor nucleic acid of the present invention or chromosomal sequencesgenetically linked by using RFLP analysis. Linked chromosomal sequencesare within 50 centiMorgans (50 cM), within 40 or 30 cM, preferablywithin 20 or 10 cM, more preferably within 5, 3, 2, or 1 cM of thenucleic acid of the invention.

III. Traits of Interest

The present invention encompasses the identification and isolation ofcDNAs encoding genes of interest in the expression of abiotic stresstolerance. Abiotic stresses such as, but not limited to, cold, heat,drought or salt stress can significantly affect the growth and/or yieldof plants. Altering the expression of genes related to these traits canbe used to improve or modify the rice plants and/or grain, as desired.Examples describe the isolated genes of interest and methods ofanalyzing the alteration of expression and their effects on the plantcharacteristics.

One aspect of the present invention provides compositions and methodsfor altering (i.e. increasing or decreasing) the level of nucleic acidmolecules and polypeptides of the present invention in plants. Inparticular, the nucleic acid molecules and polypeptides of the inventionare expressed constitutively, temporally or spatially, e.g. atdevelopmental stages, in certain tissues, and/or quantities, which areuncharacteristic of non-recombinantly engineered plants. Therefore, thepresent invention provides utility in such exemplary applications asaltering the specified characteristics identified above.

VI. Controlling Gene Expression in Transgenic Plants

The invention further relates to transformed cells comprising thenucleic acid molecules, transformed plants, seeds, and plant parts, andmethods of modifying phenotypic traits of interest by altering theexpression of the genes of the invention.

A. Modification of Coding Sequences and Adjacent Sequences

The transgenic expression in plants of genes derived from heterologoussources may involve the modification of those genes to achieve andoptimize their expression in plants. In particular, bacterial ORFs whichencode separate enzymes but which are encoded by the same transcript inthe native microbe are best expressed in plants on separate transcripts.To achieve this, each microbial ORF is isolated individually and clonedwithin a cassette which provides a plant promoter sequence at the 5′ endof the ORF and a plant transcriptional terminator at the 3′ end of theORF. The isolated ORF sequence preferably includes the initiating ATGcodon and the terminating STOP codon but may include additional sequencebeyond the initiating ATG and the STOP codon. In addition, the ORF maybe truncated, but still retain the required activity; for particularlylong ORFs, truncated versions which retain activity may be preferablefor expression in transgenic organisms. By “plant promoter” and “planttranscriptional terminator” it is intended to mean promoters andtranscriptional terminators that operate within plant cells. Thisincludes promoters and transcription terminators that may be derivedfrom non-plant sources such as viruses (an example is the CauliflowerMosaic Virus).

In some cases, modification to the ORF coding sequences and adjacentsequence is not required. It is sufficient to isolate a fragmentcontaining the ORF of interest and to insert it downstream of a plantpromoter. For example, Gaffney et al. (Science 261: 754–756 (1993)) haveexpressed the Pseudomonas nahG gene in transgenic plants under thecontrol of the CaMV 35S promoter and the CaMV tml terminatorsuccessfully without modification of the coding sequence and withnucleotides of the Pseudomonas gene upstream of the ATG still attached,and nucleotides downstream of the STOP codon still attached to the nahGORF. Preferably, as little adjacent microbial sequence as possibleshould be left attached upstream of the ATG and downstream of the STOPcodon. In practice, such construction may depend on the availability ofrestriction sites.

In other cases, the expression of genes derived from microbial sourcesmay provide problems in expression. These problems have been wellcharacterized in the art and are particularly common with genes derivedfrom certain sources such as Bacillus. These problems may apply to thenucleotide sequence of this invention and the modification of thesegenes can be undertaken using techniques now well known in the art. Thefollowing problems may be encountered:

1. Codon Usage.

The preferred codon usage in plants differs from the preferred codonusage in certain microorganisms. Comparison of the usage of codonswithin a cloned microbial ORF to usage in plant genes (and in particulargenes from the target plant) will enable an identification of the codonswithin the ORF that should preferably be changed. Typically plantevolution has tended towards a strong preference of the nucleotides Cand G in the third base position of monocotyledons, whereas dicotyledonsoften use the nucleotides A or T at this position. By modifying a geneto incorporate preferred codon usage for a particular target transgenicspecies, many of the problems described below for GC/AT content andillegitimate splicing will be overcome.

2. GC/AT Content.

Plant genes typically have a GC content of more than 35%. ORF sequenceswhich are rich in A and T nucleotides can cause several problems inplants. Firstly, motifs of ATTTA are believed to cause destabilizationof messages and are found at the 3′ end of many short-lived mRNAs.Secondly, the occurrence of polyadenylation signals such as AATAAA atinappropriate positions within the message is believed to causepremature truncation of transcription. In addition, monocotyledons mayrecognize AT-rich sequences as splice sites (see below).

3. Sequences Adjacent to the Initiating Methionine.

Plants differ from microorganisms in that their messages do not possessa defined ribosome-binding site. Rather, it is believed that ribosomesattach to the 5′ end of the message and scan for the first available ATGat which to start translation. Nevertheless, it is believed that thereis a preference for certain nucleotides adjacent to the ATG and thatexpression of microbial genes can be enhanced by the inclusion of aeukaryotic consensus translation initiator at the ATG. Clontech(1993/1994 catalog, page 210, incorporated herein by reference) havesuggested one sequence as a consensus translation initiator for theexpression of the E. coli uidA gene in plants. Further, Joshi (N.A.R.15: 6643–6653 (1987), incorporated herein by reference) has comparedmany plant sequences adjacent to the ATG and suggests another consensussequence. In situations where difficulties are encountered in theexpression of microbial ORFs in plants, inclusion of one of thesesequences at the initiating ATG may improve translation. In such casesthe last three nucleotides of the consensus may not be appropriate forinclusion in the modified sequence due to their modification of thesecond AA residue. Preferred sequences adjacent to the initiatingmethionine may differ between different plant species. A survey of 14maize genes located in the GenBank database provided the followingresults:

Position Before the Initiating ATG in 14 Maize Genes:

−10 −9 −8 −7 −6 −5 −4 −3 −2 −1 C 3 8 4 6 2 5 6 0 10 7 T 3 0 3 4 3 2 1 11 0 A 2 3 1 4 3 2 3 7 2 3 G 6 3 6 0 6 5 4 6 1 5This analysis can be done for the desired plant species into which thenucleotide sequence is being incorporated, and the sequence adjacent tothe ATG modified to incorporate the preferred nucleotides.

4. Removal of Illegitimate Splice Sites.

Genes cloned from non-plant sources and not optimized for expression inplants may also contain motifs which may be recognized in plants as 5′or 3′ splice sites, and be cleaved, thus generating truncated or deletedmessages. These sites can be removed using the techniques well known inthe art.

Techniques for the modification of coding sequences and adjacentsequences are well known in the art. In cases where the initialexpression of a microbial ORF is low and it is deemed appropriate tomake alterations to the sequence as described above, then theconstruction of synthetic genes can be accomplished according to methodswell known in the art. These are, for example, described in thepublished patent disclosures EP 0 385 962 (to Monsanto), EP 0 359 472(to Lubrizol) and WO 93/07278 (to Ciba-Geigy), all of which areincorporated herein by reference. In most cases it is preferable toassay the expression of gene constructions using transient assayprotocols (which are well known in the art) prior to their transfer totransgenic plants.

B. Construction of Plant Expression Cassettes

Coding sequences intended for expression in transgenic plants are firstassembled in expression cassettes behind a suitable promoter expressiblein plants. The expression cassettes may also comprise any furthersequences required or selected for the expression of the transgene. Suchsequences include, but are not restricted to, transcription terminators,extraneous sequences to enhance expression such as introns, vitalsequences, and sequences intended for the targeting of the gene productto specific organelles and cell compartments. These expression cassettescan then be easily transferred to the plant transformation vectorsdescribed below. The following is a description of various components oftypical expression cassettes.

1. Promoters

The selection of the promoter used in expression cassettes willdetermine the spatial and temporal expression pattern of the transgenein the transgenic plant. Selected promoters will express transgenes inspecific cell types (such as leaf epidermal cells, mesophyll cells, rootcortex cells) or in specific tissues or organs (roots, leaves orflowers, for example) and the selection will reflect the desiredlocation of accumulation of the gene product. Alternatively, theselected promoter may drive expression of the gene under variousinducing conditions. Promoters vary in their strength, i.e., ability topromote transcription. Depending upon the host cell system utilized, anyone of a number of suitable promoters can be used, including the gene'snative promoter. The following are non-limiting examples of promotersthat may be used in expression cassettes.

a. Constitutive Expression, the Ubiquitin Promoter:

Ubiquitin is a gene product known to accumulate in many cell types andits promoter has been cloned from several species for use in transgenicplants (e.g. sunflower—Binet et al. Plant Science 79: 87–94 (1991);maize—Christensen et al. Plant Molec. Biol. 12: 619–632 (1989); andArabidopsis—Callis et al., J. Biol. Chem. 265:12486–12493 (1990) andNorris et al., Plant Mol. Biol. 21:895–906 (1993)). The maize ubiquitinpromoter has been developed in transgenic monocot systems and itssequence and vectors constructed for monocot transformation aredisclosed in the patent publication EP 0 342 926 (to Lubrizol) which isherein incorporated by reference. Taylor et al. (Plant Cell Rep. 12:491–495 (1993)) describe a vector (pAHC25) that comprises the maizeubiquitin promoter and first intron and its high activity in cellsuspensions of numerous monocotyledons when introduced viamicroprojectile bombardment. The Arabidopsis ubiquitin promoter is idealfor use with the nucleotide sequences of the present invention. Theubiquitin promoter is suitable for gene expression in transgenic plants,both monocotyledons and dicotyledons. Suitable vectors are derivativesof pAHC25 or any of the transformation vectors described in thisapplication, modified by the introduction of the appropriate ubiquitinpromoter and/or intron sequences.

b. Constitutive Expression, the CaMV 35S Promoter:

Construction of the plasmid pCGN1761 is described in the publishedpatent application EP 0 392 225 (Example 23), which is herebyincorporated by reference. pCGN1761 contains the “double” CaMV 35Spromoter and the tml transcriptional terminator with a unique EcoRI sitebetween the promoter and the terminator and has a pUC-type backbone. Aderivative of pCGN1761 is constructed which has a modified polylinkerwhich includes NotI and XhoI sites in addition to the existing EcoRIsite. This derivative is designated pCGN1761ENX. pCGN1761ENX is usefulfor the cloning of cDNA sequences or coding sequences (includingmicrobial ORF sequences) within its polylinker for the purpose of theirexpression under the control of the 35S promoter in transgenic plants.The entire 35S promoter-coding sequence-tml terminator cassette of sucha construction can be excised by HindIII, SphI, SalI, and XbaI sites 5′to the promoter and XbaI, BamHI and BglI sites 3′ to the terminator fortransfer to transformation vectors such as those described below.Furthermore, the double 35S promoter fragment can be removed by 5′excision with HindIII, SphI, SalI, XbaI, or PstI, and 3′ excision withany of the polylinker restriction sites (EcoRI, NotI or XhoI) forreplacement with another promoter. If desired, modifications around thecloning sites can be made by the introduction of sequences that mayenhance translation. This is particularly useful when overexpression isdesired. For example, pCGN1761ENX may be modified by optimization of thetranslational initiation site as described in Example 37 of U.S. Pat.No. 5,639,949, incorporated herein by reference.

c. Constitutive Expression, the Actin Promoter:

Several isoforms of actin are known to be expressed in most cell typesand consequently the actin promoter is a good choice for a constitutivepromoter. In particular, the promoter from the rice ActI gene has beencloned and characterized (McElroy et al. Plant Cell 2: 163–171 (1990)).A 1.3 kb fragment of the promoter was found to contain all theregulatory elements required for expression in rice protoplasts.Furthermore, numerous expression vectors based on the ActI promoter havebeen constructed specifically for use in monocotyledons (McElroy et al.Mol. Gen. Genet. 231: 150–160 (1991)). These incorporate the ActI-intron1, AdhI 5′ flanking sequence and AdhI-intron 1 (from the maize alcoholdehydrogenase gene) and sequence from the CaMV 35S promoter. Vectorsshowing highest expression were fusions of 35S and ActI intron or theActI 5′ flanking sequence and the ActI intron. Optimization of sequencesaround the initiating ATG (of the GUS reporter gene) also enhancedexpression. The promoter expression cassettes described by McElroy etal. (Mol. Gen. Genet. 231: 150–160 (1991)) can be easily modified forgene expression and are particularly suitable for use inmonocotyledonous hosts. For example, promoter-containing fragments isremoved from the McElroy constructions and used to replace the double35S promoter in pCGN1761ENX, which is then available for the insertionof specific gene sequences. The fusion genes thus constructed can thenbe transferred to appropriate transformation vectors. In a separatereport, the rice ActI promoter with its first intron has also been foundto direct high expression in cultured barley cells (Chibbar et al. PlantCell Rep. 12: 506–509 (1993)).

d. Seed-specific Expression

Seed specific expression can be obtained by utilizing expression vectorsor cassettes with seed-specific promoters such as but not limited to,the ADPgpp, gamma-zein, glutelin, RS-4, globulin, or oleosin promoter.

e. Inducible Expression, PR-1 Promoters:

The double 35S promoter in pCGN1761ENX may be replaced with any otherpromoter of choice that will result in suitably high expression levels.By way of example, one of the chemically regulatable promoters describedin U.S. Pat. No. 5,614,395, such as the tobacco PR-1a promoter, mayreplace the double 35S promoter. Alternately, the Arabidopsis PR-1promoter described in Lebel et al., Plant J. 16:223–233 (1998) may beused. The promoter of choice is preferably excised from its source byrestriction enzymes, but can alternatively be PCR-amplified usingprimers that carry appropriate terminal restriction sites. ShouldPCR-amplification be undertaken, the promoter should be re-sequenced tocheck for amplification errors after the cloning of the amplifiedpromoter in the target vector. The chemically/pathogen regulatabletobacco PR-1a promoter is cleaved from plasmid pCIB1004 (forconstruction, see example 21 of EP 0 332 104, which is herebyincorporated by reference) and transferred to plasmid pCGN1761ENX (Ukneset al., Plant Cell 4: 645–656 (1992)). pCIB1004 is cleaved with NcoI andthe resultant 3′ overhang of the linearized fragment is rendered bluntby treatment with T4 DNA polymerase. The fragment is then cleaved withHindIII and the resultant PR-1a promoter-containing fragment is gelpurified and cloned into pCGN1761ENX from which the double 35S promoterhas been removed. This is accomplished by cleavage with XhoI andblunting with T4 polymerase, followed by cleavage with HindIII, andisolation of the larger vector-terminator containing fragment into whichthe pCIB1004 promoter fragment is cloned. This generates a pCGN1761ENXderivative with the PR-1a promoter and the tml terminator and anintervening polylinker with unique EcoRI and NotI sites. The selectedcoding sequence can be inserted into this vector, and the fusionproducts (i.e. promoter-gene-terminator) can subsequently be transferredto any selected transformation vector, including those described infra.Various chemical regulators may be employed to induce expression of theselected coding sequence in the plants transformed according to thepresent invention, including the benzothiadiazole, isonicotinic acid,and salicylic acid compounds disclosed in U.S. Pat. Nos. 5,523,311 and5,614,395.

e. Inducible Expression, an Ethanol-Inducible Promoter:

A promoter inducible by certain alcohols or ketones, such as ethanol,may also be used to confer inducible expression of a coding sequence ofthe present invention. Such a promoter is for example the alcA genepromoter from Aspergillus nidulans (Caddick et al. (1998) Nat.Biotechnol 16:177–180). In A. nidulans, the alcA gene encodes alcoholdehydrogenase I, the expression of which is regulated by the AlcRtranscription factors in presence of the chemical inducer. For thepurposes of the present invention, the CAT coding sequences in plasmidpalcA:CAT comprising a alcA gene promoter sequence fused to a minimal35S promoter (Caddick et al. (1998) Nat. Biotechnol 16:177–180) arereplaced by a coding sequence of the present invention to form anexpression cassette having the coding sequence under the control of thealcA gene promoter. This is carried out using methods well known in theart.

f. Inducible Expression, a Glucocorticoid-Inducible Promoter:

Induction of expression of a nucleic acid sequence of the presentinvention using systems based on steroid hormones is also contemplated.For example, a glucocorticoid-mediated induction system is used (Aoyamaand Chua (1997) The Plant Journal 11: 605–612) and gene expression isinduced by application of a glucocorticoid, for example a syntheticglucocorticoid, preferably dexamethasone, preferably at a concentrationranging from 0.1 mM to 1 mM, more preferably from 10 mM to 100 mM. Forthe purposes of the present invention, the luciferase gene sequences arereplaced by a nucleic acid sequence of the invention to form anexpression cassette having a nucleic acid sequence of the inventionunder the control of six copies of the GAL4 upstream activatingsequences fused to the 35S minimal promoter. This is carried out usingmethods well known in the art. The trans-acting factor comprises theGAL4 DNA-binding domain (Keegan et al. (1986) Science 231: 699–704)fused to the transactivating domain of the herpes viral protein VP16(Triezenberg et al. (1988) Genes Devel. 2: 718–729) fused to thehormone-binding domain of the rat glucocorticoid receptor (Picard et al.(1988) Cell 54: 1073–1080). The expression of the fusion protein iscontrolled either by a promoter known in the art or described here. Thisexpression cassette is also comprised in the plant comprising a nucleicacid sequence of the invention fused to the 6xGAL4/minimal promoter.Thus, tissue- or organ-specificity of the fusion protein is achievedleading to inducible tissue- or organ-specificity of the insecticidaltoxin.

g. Root Specific Expression:

Another pattern of gene expression is root expression. A suitable rootpromoter is the promoter of the maize metallothionein-like (MTL) genedescribed by de Framond (FEBS 290: 103–106 (1991)) and also in U.S. Pat.No. 5,466,785, incorporated herein by reference. This “MTL” promoter istransferred to a suitable vector such as pCGN1761ENX for the insertionof a selected gene and subsequent transfer of the entirepromoter-gene-terminator cassette to a transformation vector ofinterest.

h. Wound-Inducible Promoters:

Wound-inducible promoters may also be suitable for gene expression.Numerous such promoters have been described (e.g. Xu et al. Plant Molec.Biol. 22: 573–588 (1993), Logemann et al. Plant Cell 1: 151–158 (1989),Rohrmeier & Lehle, Plant Molec. Biol. 22: 783–792 (1993), Firek et al.Plant Molec. Biol. 22: 129–142 (1993), Warner et al. Plant J. 3: 191–201(1993)) and all are suitable for use with the instant invention.Logemann et al. describe the 5′ upstream sequences of the dicotyledonouspotato wunI gene. Xu et al. show that a wound-inducible promoter fromthe dicotyledon potato (pin2) is active in the monocotyledon rice.Further, Rohrmeier & Lehle describe the cloning of the maize WipI cDNAwhich is wound induced and which can be used to isolate the cognatepromoter using standard techniques. Similar, Firek et al. and Warner etal. have described a wound-induced gene from the monocotyledon Asparagusofficinalis, which is expressed at local wound and pathogen invasionsites. Using cloning techniques well known in the art, these promoterscan be transferred to suitable vectors, fused to the genes pertaining tothis invention, and used to express these genes at the sites of plantwounding.

i. Pith-Preferred Expression:

Patent Application WO 93/07278, which is herein incorporated byreference, describes the isolation of the maize trpA gene, which ispreferentially expressed in pith cells. The gene sequence and promoterextending up to −1726 bp from the start of transcription are presented.Using standard molecular biological techniques, this promoter, or partsthereof, can be transferred to a vector such as pCGN1761 where it canreplace the 35S promoter and be used to drive the expression of aforeign gene in a pith-preferred manner. In fact, fragments containingthe pith-preferred promoter or parts thereof can be transferred to anyvector and modified for utility in transgenic plants.

j. Leaf-Specific Expression:

A maize gene encoding phosphoenol carboxylase (PEPC) has been describedby Hudspeth & Grula (Plant Molec Biol 12: 579–589 (1989)). Usingstandard molecular biological techniques the promoter for this gene canbe used to drive the expression of any gene in a leaf-specific manner intransgenic plants.

k. Pollen-Specific Expression:

WO 93/07278 describes the isolation of the maize calcium-dependentprotein kinase (CDPK) gene which is expressed in pollen cells. The genesequence and promoter extend up to 1400 bp from the start oftranscription. Using standard molecular biological techniques, thispromoter or parts thereof, can be transferred to a vector such aspCGN1761 where it can replace the 35S promoter and be used to drive theexpression of a nucleic acid sequence of the invention in apollen-specific manner.

2. Transcriptional Terminators

A variety of transcriptional terminators are available for use inexpression cassettes. These are responsible for the termination oftranscription beyond the transgene and correct mRNA polyadenylation.Appropriate transcriptional terminators are those that are known tofunction in plants and include the CaMV 35S terminator, the tmlterminator, the nopaline synthase terminator and the pea rbcS E9terminator. These can be used in both monocotyledons and dicotyledons.In addition, a gene's native transcription terminator may be used.

3. Sequences for the Enhancement or Regulation of Expression

Numerous sequences have been found to enhance gene expression fromwithin the transcriptional unit and these sequences can be used inconjunction with the genes of this invention to increase theirexpression in transgenic plants.

Various intron sequences have been shown to enhance expression,particularly in monocotyledonous cells. For example, the introns of themaize AdhI gene have been found to significantly enhance the expressionof the wild-type gene under its cognate promoter when introduced intomaize cells. Intron 1 was found to be particularly effective andenhanced expression in fusion constructs with the chloramphenicolacetyltransferase gene (Callis et al., Genes Develop. 1: 1183–1200(1987)). In the same experimental system, the intron from the maizebronze1 gene had a similar effect in enhancing expression. Intronsequences have been routinely incorporated into plant transformationvectors, typically within the non-translated leader.

A number of non-translated leader sequences derived from viruses arealso known to enhance expression, and these are particularly effectivein dicotyledonous cells. Specifically, leader sequences from TobaccoMosaic Virus (TMV, the “W-sequence”), Maize Chlorotic Mottle Virus(MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effectivein enhancing expression (e.g. Gallie et al. Nucl. Acids Res. 15:8693–8711 (1987); Skuzeski et al. Plant Molec. Biol. 15: 65–79 (1990)).Other leader sequences known in the art include but are not limited to:picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′noncoding region) (Elroy-Stein, O., Fuerst, T. R., and Moss, B. PNAS USA86:6126–6130 (1989)); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Allison et al., 1986); MDMV leader (Maize DwarfMosaic Virus); Virology 154:9–20); human immunoglobulin heavy-chainbinding protein (BiP) leader, (Macejak, D. G., and Sarnow, P., Nature353: 90–94 (1991); untranslated leader from the coat protein mRNA ofalfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., and Gehrke, L.,Nature 325:622–625 (1987); tobacco mosaic virus leader (TMV), (Gallie,D. R. et al., Molecular Biology of RNA, pages 237–256 (1989); and MaizeChlorotic Mottle Virus leader (MCMV) (Lommel, S. A. et al., Virology81:382–385 (1991). See also, Della-Cioppa et al., Plant Physiology84:965–968 (1987).

In addition to incorporating one or more of the aforementioned elementsinto the 5′ regulatory region of a target expression cassette of theinvention, other elements peculiar to the target expression cassette mayalso be incorporated. Such elements include but are not limited to aminimal promoter. By minimal promoter it is intended that the basalpromoter elements are inactive or nearly so without upstream activation.Such a promoter has low background activity in plants when there is notransactivator present or when enhancer or response element bindingsites are absent. One minimal promoter that is particularly useful fortarget genes in plants is the Bz1 minimal promoter, which is obtainedfrom the bronze1 gene of maize. The Bz1 core promoter is obtained fromthe “myc” mutant Bz1-luciferase construct pBz1LucR98 via cleavage at theNheI site located at −53 to −58. Roth et al., Plant Cell 3: 317 (1991).The derived Bz1 core promoter fragment thus extends from −53 to +227 andincludes the Bz1 intron-1 in the 5′ untranslated region. Also useful forthe invention is a minimal promoter created by use of a synthetic TATAelement. The TATA element allows recognition of the promoter by RNApolymerase factors and confers a basal level of gene expression in theabsence of activation (see generally, Mukumoto (1993) Plant Mol Biol 23:995–1003; Green (2000) Trends Biochem Sci 25: 59–63)

4. Targeting of the Gene Product Within the Cell

Various mechanisms for targeting gene products are known to exist inplants and the sequences controlling the functioning of these mechanismshave been characterized in some detail. For example, the targeting ofgene products to the chloroplast is controlled by a signal sequencefound at the amino terminal end of various proteins that is cleavedduring chloroplast import to yield the mature protein (e.g. Comai et al.J. Biol. Chem. 263: 15104–15109 (1988)). These signal sequences can befused to heterologous gene products to effect the import of heterologousproducts into the chloroplast (van den Broeck, et al. Nature 313:358–363 (1985)). DNA encoding for appropriate signal sequences can beisolated from the 5′ end of the cDNAs encoding the RUBISCO protein, theCAB protein, the EPSP synthase enzyme, the GS2 protein and many otherproteins which are known to be chloroplast localized. See also, thesection entitled “Expression With Chloroplast Targeting” in Example 37of U.S. Pat. No. 5,639,949.

Other gene products are localized to other organelles such as themitochondrion and the peroxisome (e.g. Unger et al. Plant Molec. Biol.13: 411–418 (1989)). The cDNAs encoding these products can also bemanipulated to effect the targeting of heterologous gene products tothese organelles. Examples of such sequences are the nuclear-encodedATPases and specific aspartate amino transferase isoforms formitochondria. Targeting cellular protein bodies has been described byRogers et al. (Proc. Natl. Acad. Sci. USA 82: 6512–6516 (1985)).

In addition, sequences have been characterized which cause the targetingof gene products to other cell compartments. Amino terminal sequencesare responsible for targeting to the ER, the apoplast, and extracellularsecretion from aleurone cells (Koehler & Ho, Plant Cell 2: 769–783(1990)). Additionally, amino terminal sequences in conjunction withcarboxy terminal sequences are responsible for vacuolar targeting ofgene products (Shinshi et al. Plant Molec. Biol. 14: 357–368 (1990)).

By the fusion of the appropriate targeting sequences described above totransgene sequences of interest it is possible to direct the transgeneproduct to any organelle or cell compartment. For chloroplast targeting,for example, the chloroplast signal sequence from the RUBISCO gene, theCAB gene, the EPSP synthase gene, or the GS2 gene is fused in frame tothe amino terminal ATG of the transgene. The signal sequence selectedshould include the known cleavage site, and the fusion constructedshould take into account any amino acids after the cleavage site whichare required for cleavage. In some cases this requirement may befulfilled by the addition of a small number of amino acids between thecleavage site and the transgene ATG or, alternatively, replacement ofsome amino acids within the transgene sequence. Fusions constructed forchloroplast import can be tested for efficacy of chloroplast uptake byin vitro translation of in vitro transcribed constructions followed byin vitro chloroplast uptake using techniques described by Bartlett etal. In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology,Elsevier pp 1081–1091 (1982) and Wasmann et al. Mol. Gen. Genet. 205:446–453 (1986). These construction techniques are well known in the artand are equally applicable to mitochondria and peroxisomes.

The above-described mechanisms for cellular targeting can be utilizednot only in conjunction with their cognate promoters, but also inconjunction with heterologous promoters so as to effect a specificcell-targeting goal under the transcriptional regulation of a promoterthat has an expression pattern different to that of the promoter fromwhich the targeting signal derives.

C. Construction of Plant Transformation Vectors

Numerous transformation vectors available for plant transformation areknown to those of ordinary skill in the plant transformation arts, andthe genes pertinent to this invention can be used in conjunction withany such vectors. The selection of vector will depend upon the preferredtransformation technique and the target species for transformation. Forcertain target species, different antibiotic or herbicide selectionmarkers may be preferred. Selection markers used routinely intransformation include the nptlI gene, which confers resistance tokanamycin and related antibiotics (Messing & Vierra. Gene 19: 259–268(1982); Bevan et al., Nature 304:184–187 (1983)), the bar gene, whichconfers resistance to the herbicide phosphinothricin (White et al.,Nucl. Acids Res 18: 1062 (1990), Spencer et al. Theor. Appl. Genet 79:625–631 (1990)), the hph gene, which confers resistance to theantibiotic hygromycin (Blochinger & Diggelmann, Mol Cell Biol 4:2929–2931), and the dhfr gene, which confers resistance to methatrexate(Bourouis et al., EMBO J. 2(7): 1099–1104 (1983)), the EPSPS gene, whichconfers resistance to glyphosate (U.S. Pat. Nos. 4,940,935 and5,188,642), and the mannose-6-phosphate isomerase gene, which providesthe ability to metabolize mannose (U.S. Pat. Nos. 5,767,378 and5,994,629).

1. Vectors Suitable for Agrobacterium Transformation

Many vectors are available for transformation using Agrobacteriumtumefaciens. These typically carry at least one T-DNA border sequenceand include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)).Below, the construction of two typical vectors suitable forAgrobacterium transformation is described.

a. pCIB200 and pCIB2001:

The binary vectors pCIB200 and pCIB2001 are used for the construction ofrecombinant vectors for use with Agrobacterium and are constructed inthe following manner. pTJS75kan is created by Narl digestion of pTJS75(Schmidhauser & Helinski, J. Bacteriol. 164: 446–455 (1985)) allowingexcision of the tetracycline-resistance gene, followed by insertion ofan AccI fragment from pUC4K carrying an NPTII (Messing & Vierra, Gene19: 259–268 (1982): Bevan et al., Nature 304: 184–187 (1983): McBride etal., Plant Molecular Biology 14: 266–276 (1990)). XhoI linkers areligated to the EcoRV fragment of PCIB7 which contains the left and rightT-DNA borders, a plant selectable nos/nptlI chimeric gene and the pUCpolylinker (Rothstein et al., Gene 53: 153–161 (1987)), and theXhoI-digested fragment are cloned into SalI-digested pTJS75kan to createpCIB200 (see also EP 0 332 104, example 19). pCIB200 contains thefollowing unique polylinker restriction sites: EcoRI, SstI, KpnI, BglII,XbaI, and SalI. pCIB2001 is a derivative of pCIB200 created by theinsertion into the polylinker of additional restriction sites. Uniquerestriction sites in the polylinker of pCIB2001 are EcoRI, SstI, KpnI,BglII, XbaI, SalI, MluI, BclI, AvrlI, ApaI, HpaI, and StuI. pCIB2001, inaddition to containing these unique restriction sites also has plant andbacterial kanamycin selection, left and right T-DNA borders forAgrobacterium-mediated transformation, the RK2-derived trfA function formobilization between E. coli and other hosts, and the OriT and OriVfunctions also from RK2. The pCIB2001 polylinker is suitable for thecloning of plant expression cassettes containing their own regulatorysignals.

b. pCIB10 and Hygromycin Selection Derivatives Thereof:

The binary vector pCIB10 contains a gene encoding kanamycin resistancefor selection in plants and T-DNA right and left border sequences andincorporates sequences from the wide host-range plasmid pRK252 allowingit to replicate in both E. coli and Agrobacterium. Its construction isdescribed by Rothstein et al. (Gene 53: 153–161 (1987)). Variousderivatives of pCIB10 are constructed which incorporate the gene forhygromycin B phosphotransferase described by Gritz et al. (Gene 25:179–188 (1983)). These derivatives enable selection of transgenic plantcells on hygromycin only (pCIB743), or hygromycin and kanamycin(pCIB715, pCIB717).

2. Vectors Suitable for Non-Agrobacterium Transformation

Transformation without the use of Agrobacterium tumefaciens circumventsthe requirement for T-DNA sequences in the chosen transformation vectorand consequently vectors lacking these sequences can be utilized inaddition to vectors such as the ones described above which contain T-DNAsequences. Transformation techniques that do not rely on Agrobacteriuminclude transformation via particle bombardment, protoplast uptake (e.g.PEG and electroporation) and microinjection. The choice of vectordepends largely on the preferred selection for the species beingtransformed. Below, the construction of typical vectors suitable fornon-Agrobacterium transformation is described.

a. pCIB3064:

pCIB3064 is a pUC-derived vector suitable for direct gene transfertechniques in combination with selection by the herbicide basta (orphosphinothricin). The plasmid pCIB246 comprises the CaMV 35S promoterin operational fusion to the E. coli GUS gene and the CaMV 35Stranscriptional terminator and is described in the PCT publishedapplication WO 93/07278. The 35S promoter of this vector contains twoATG sequences 5′ of the start site. These sites are mutated usingstandard PCR techniques in such a way as to remove the ATGs and generatethe restriction sites SspI and PvulI. The new restriction sites are 96and 37 bp away from the unique SalI site and 101 and 42 bp away from theactual start site. The resultant derivative of pCIB246 is designatedpCIB3025. The GUS gene is then excised from pCIB3025 by digestion withSalI and SacI, the termini rendered blunt and religated to generateplasmid pCIB3060. The plasmid pJIT82 is obtained from the John InnesCentre, Norwich and the a 400 bp SmaI fragment containing the bar genefrom Streptomyces viridochromogenes is excised and inserted into theHpaI site of pCIB3060 (Thompson et al. EMBO J 6: 2519–2523 (1987)). Thisgenerated pCIB3064, which comprises the bar gene under the control ofthe CaMV 35S promoter and terminator for herbicide selection, a gene forampicillin resistance (for selection in E. coli) and a polylinker withthe unique sites SphI, PstI, HindIII, and BamHI. This vector is suitablefor the cloning of plant expression cassettes containing their ownregulatory signals.

b. pSOG19 and pSOG35:

pSOG35 is a transformation vector that utilizes the E. coli genedihydrofolate reductase (DFR) as a selectable marker conferringresistance to methotrexate. PCR is used to amplify the 35S promoter(−800 bp), intron 6 from the maize Adh1 gene (−550 bp) and 18 bp of theGUS untranslated leader sequence from pSOG10. A 250-bp fragment encodingthe E. coli dihydrofolate reductase type II gene is also amplified byPCR and these two PCR fragments are assembled with a SacI-PstI fragmentfrom pB1221 (Clontech) which comprises the pUC19 vector backbone and thenopaline synthase terminator. Assembly of these fragments generatespSOG19 which contains the 35S promoter in fusion with the intron 6sequence, the GUS leader, the DHFR gene and the nopaline synthaseterminator. Replacement of the GUS leader in pSOG19 with the leadersequence from Maize Chlorotic Mottle Virus (MCMV) generates the vectorpSOG35. pSOG19 and pSOG35 carry the pUC gene for ampicillin resistanceand have HindIII, SphI, PstI and EcoRI sites available for the cloningof foreign substances.

3. Vector Suitable for Chloroplast Transformation

For expression of a nucleotide sequence of the present invention inplant plastids, plastid transformation vector pPH143 (WO 97/32011,example 36) is used. The nucleotide sequence is inserted into pPH143thereby replacing the PROTOX coding sequence. This vector is then usedfor plastid transformation and selection of transformants forspectinomycin resistance. Alternatively, the nucleotide sequence isinserted in pPH143 so that it replaces the aadH gene. In this case,transformants are selected for resistance to PROTOX inhibitors.

D. Transformation

Once a nucleic acid sequence of the invention has been cloned into anexpression system, it is transformed into a plant cell. The receptor andtarget expression cassettes of the present invention can be introducedinto the plant cell in a number of art-recognized ways. Methods forregeneration of plants are also well known in the art. For example, Tiplasmid vectors have been utilized for the delivery of foreign DNA, aswell as direct DNA uptake, liposomes, electroporation, microinjection,and microprojectiles. In addition, bacteria from the genus Agrobacteriumcan be utilized to transform plant cells. Below are descriptions ofrepresentative techniques for transforming both dicotyledonous andmonocotyledonous plants, as well as a representative plastidtransformation technique.

1. Transformation of Dicotyledons

Transformation techniques for dicotyledons are well known in the art andinclude Agrobacterium-based techniques and techniques that do notrequire Agrobacterium. Non-Agrobacterium techniques involve the uptakeof exogenous genetic material directly by protoplasts or cells. This canbe accomplished by PEG or electroporation mediated uptake, particlebombardment-mediated delivery, or microinjection. Examples of thesetechniques are described by Paszkowski et al., EMBO J 3: 2717–2722(1984), Potrykus et al., Mol. Gen. Genet. 199: 169–177 (1985), Reich etal., Biotechnology 4: 1001–1004 (1986), and Klein et al., Nature 327:70–73 (1987). In each case the transformed cells are regenerated towhole plants using standard techniques known in the art.

Agrobacterium-mediated transformation is a preferred technique fortransformation of dicotyledons because of its high efficiency oftransformation and its broad utility with many different species.Agrobacterium transformation typically involves the transfer of thebinary vector carrying the foreign DNA of interest (e.g. pCIB200 orpCIB2001) to an appropriate Agrobacterium strain which may depend of thecomplement of vir genes carried by the host Agrobacterium strain eitheron a co-resident Ti plasmid or chromosomally (e.g. strain CIB542 forpCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159–169 (1993)). Thetransfer of the recombinant binary vector to Agrobacterium isaccomplished by a triparental mating procedure using E. coli carryingthe recombinant binary vector, a helper E. coli strain which carries aplasmid such as pRK2013 and which is able to mobilize the recombinantbinary vector to the target Agrobacterium strain. Alternatively, therecombinant binary vector can be transferred to Agrobacterium by DNAtransformation (Höfgen & Willmitzer, Nucl. Acids Res. 16: 9877 (1988)).

Transformation of the target plant species by recombinant Agrobacteriumusually involves co-cultivation of the Agrobacterium with explants fromthe plant and follows protocols well known in the art. Transformedtissue is regenerated on selectable medium carrying the antibiotic orherbicide resistance marker present between the binary plasmid T-DNAborders.

Another approach to transforming plant cells with a gene involvespropelling inert or biologically active particles at plant tissues andcells. This technique is disclosed in U.S. Pat. Nos. 4,945,050,5,036,006, and 5,100,792 all to Sanford et al. Generally, this procedureinvolves propelling inert or biologically active particles at the cellsunder conditions effective to penetrate the outer surface of the celland afford incorporation within the interior thereof. When inertparticles are utilized, the vector can be introduced into the cell bycoating the particles with the vector containing the desired gene.Alternatively, the target cell can be surrounded by the vector so thatthe vector is carried into the cell by the wake of the particle.Biologically active particles (e.g., dried yeast cells, dried bacteriumor a bacteriophage, each containing DNA sought to be introduced) canalso be propelled into plant cell tissue.

2. Transformation of Monocotyledons

Transformation of most monocotyledon species has now also becomeroutine. Preferred techniques include direct gene transfer intoprotoplasts using PEG or electroporation techniques, and particlebombardment into callus tissue. Transformations can be undertaken with asingle DNA species or multiple DNA species (i.e. co-transformation) andboth these techniques are suitable for use with this invention.Co-transformation may have the advantage of avoiding complete vectorconstruction and of generating transgenic plants with unlinked loci forthe gene of interest and the selectable marker, enabling the removal ofthe selectable marker in subsequent generations, should this be regardeddesirable. However, a disadvantage of the use of co-transformation isthe less than 100% frequency with which separate DNA species areintegrated into the genome (Schocher et al. Biotechnology 4: 1093–1096(1986)).

Patent Applications EP 0 292 435, EP 0 392 225, and WO 93/07278 describetechniques for the preparation of callus and protoplasts from an eliteinbred line of maize, transformation of protoplasts using PEG orelectroporation, and the regeneration of maize plants from transformedprotoplasts. Gordon-Kamm et al. (Plant Cell 2: 603–618 (1990)) and Frommet al. (Biotechnology 8: 833–839 (1990)) have published techniques fortransformation of A188-derived maize line using particle bombardment.Furthermore, WO 93/07278 and Koziel et al. (Biotechnology 11: 194–200(1993)) describe techniques for the transformation of elite inbred linesof maize by particle bombardment. This technique utilizes immature maizeembryos of 1.5–2.5 mm length excised from a maize ear 14–15 days afterpollination and a PDS-1000He Biolistics device for bombardment.

Transformation of rice can also be undertaken by direct gene transfertechniques utilizing protoplasts or particle bombardment.Protoplast-mediated transformation has been described for Japonica-typesand Indica-types (Zhang et al. Plant Cell Rep 7: 379–384 (1988);Shimamoto et al. Nature 338: 274–277 (1989); Datta et al. Biotechnology8: 736–740 (1990)). Both types are also routinely transformable usingparticle bombardment (Christou et al. Biotechnology 9: 957–962 (1991)).Furthermore, WO 93/21335 describes techniques for the transformation ofrice via electroporation.

Patent Application EP 0 332 581 describes techniques for the generation,transformation and regeneration of Pooideae protoplasts. Thesetechniques allow the transformation of Dactylis and wheat. Furthermore,wheat transformation has been described by Vasil et al. (Biotechnology10: 667–674 (1992)) using particle bombardment into cells of type Clong-term regenerable callus, and also by Vasil et al. (Biotechnology11: 1553–1558 (1993)) and Weeks et al. (Plant Physiol. 102: 1077–1084(1993)) using particle bombardment of immature embryos and immatureembryo-derived callus. A preferred technique for wheat transformation,however, involves the transformation of wheat by particle bombardment ofimmature embryos and includes either a high sucrose or a high maltosestep prior to gene delivery. Prior to bombardment, any number of embryos(0.75–1 mm in length) are plated onto MS medium with 3% sucrose(Murashiga & Skoog, Physiologia Plantarum 15: 473–497 (1962)) and 3 mg/l2,4-D for induction of somatic embryos, which is allowed to proceed inthe dark. On the chosen day of bombardment, embryos are removed from theinduction medium and placed onto the osmoticum (i.e. induction mediumwith sucrose or maltose added at the desired concentration, typically15%). The embryos are allowed to plasmolyze for 2–3 hours and are thenbombarded. Twenty embryos per target plate is typical, although notcritical. An appropriate gene-carrying plasmid (such as pCIB3064 orpSG35) is precipitated onto micrometer size gold particles usingstandard procedures. Each plate of embryos is shot with the DuPontBiolistics® helium device using a burst pressure of ˜1000 psi using astandard 80 mesh screen. After bombardment, the embryos are placed backinto the dark to recover for about 24 hours (still on osmoticum). After24 hrs, the embryos are removed from the osmoticum and placed back ontoinduction medium where they stay for about a month before regeneration.Approximately one month later the embryo explants with developingembryogenic callus are transferred to regeneration medium (MS+1 mg/literNAA, 5 mg/liter GA), further containing the appropriate selection agent(10 mg/l basta in the case of pCIB3064 and 2 mg/l methotrexate in thecase of pSOG35). After approximately one month, developed shoots aretransferred to larger sterile containers known as “GA7s” which containhalf-strength MS, 2% sucrose, and the same concentration of selectionagent.

Tranformation of monocotyledons using Agrobacterium has also beendescribed. See, WO 94/00977 and U.S. Pat. No. 5,591,616, both of whichare incorporated herein by reference. See also, Negrotto et al., PlantCell Reports 19: 798–803 (2000), incorporated herein by reference.

For this example, rice (Oryza sativa) is used for generating transgenicplants. Various rice cultivars can be used (Hiei et al., 1994, PlantJournal 6:271–282; Dong et al., 1996, Molecular Breeding 2:267–276; Hieiet al., 1997, Plant Molecular Biology, 35:205–218). Also, the variousmedia constituents described below may be either varied in quantity orsubstituted. Embryogenic responses are initiated and/or cultures areestablished from mature embryos by culturing on MS-CIM medium (MS basalsalts, 4.3 g/liter; B5 vitamins (200×), 5 ml/liter; Sucrose, 30 g/liter;proline, 500 mg/liter; glutamine, 500 mg/liter; casein hydrolysate, 300mg/liter; 2,4-D (1 mg/ml), 2 ml/liter; adjust pH to 5.8 with 1 N KOH;Phytagel, 3 g/liter). Either mature embryos at the initial stages ofculture response or established culture lines are inoculated andco-cultivated with the Agrobacterium tumefaciens strain LBA4404(Agrobacterium) containing the desired vector construction.Agrobacterium is cultured from glycerol stocks on solid YPC medium (100mg/L spectinomycin and any other appropriate antibiotic) for ˜2 days at28° C. Agrobacterium is re-suspended in liquid MS-CIM medium. TheAgrobacterium culture is diluted to an OD600 of 0.2–0.3 andacetosyringone is added to a final concentration of 200 uM.Acetosyringone is added before mixing the solution with the ricecultures to induce Agrobacterium for DNA transfer to the plant cells.For inoculation, the plant cultures are immersed in the bacterialsuspension. The liquid bacterial suspension is removed and theinoculated cultures are placed on co-cultivation medium and incubated at22° C. for two days. The cultures are then transferred to MS-CIM mediumwith Ticarcillin (400 mg/liter) to inhibit the growth of Agrobacterium.For constructs utilizing the PMI selectable marker gene (Reed et al., InVitro Cell. Dev. Biol.-Plant 37:127–132), cultures are transferred toselection medium containing Mannose as a carbohydrate source (MS with 2%Mannose, 300 mg/liter Ticarcillin) after 7 days, and cultured for 3–4weeks in the dark. Resistant colonies are then transferred toregeneration induction medium (MS with no 2,4-D, 0.5 mg/liter IAA, 1mg/liter zeatin, 200 mg/liter timentin 2% Mannose and 3% Sorbitol) andgrown in the dark for 14 days. Proliferating colonies are thentransferred to another round of regeneration induction media and movedto the light growth room. Regenerated shoots are transferred to GA7containers with GA7-1 medium (MS with no hormones and 2% Sorbitol) for 2weeks and then moved to the greenhouse when they are large enough andhave adequate roots. Plants are transplanted to soil in the greenhouse(T₀ generation) grown to maturity, and the T₁ seed is harvested.

3. Transformation of Plastids

Seeds of Nicotiana tabacum c.v. ‘Xanthi nc’ are germinated seven perplate in a 1″ circular array on T agar medium and bombarded 12–14 daysafter sowing with 1 μm tungsten particles (M10, Biorad, Hercules,Calif.) coated with DNA from plasmids pPH143 and pPH145 essentially asdescribed (Svab, Z. and Maliga, P. (1993) PNAS 90, 913–917). Bombardedseedlings are incubated on T medium for two days after which leaves areexcised and placed abaxial side up in bright light (350–500 μmolphotons/m²/s) on plates of RMOP medium (Svab, Z., Hajdukiewicz, P. andMaliga, P. (1990) PNAS 87, 8526–8530) containing 500 μg/ml spectinomycindihydrochloride (Sigma, St. Louis, Mo.). Resistant shoots appearingunderneath the bleached leaves three to eight weeks after bombardmentare subcloned onto the same selective medium, allowed to form callus,and secondary shoots isolated and subcloned. Complete segregation oftransformed plastid genome copies (homoplasmicity) in independentsubclones is assessed by standard techniques of Southern blotting(Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor). BamHI/EcoRI-digestedtotal cellular DNA (Mettler, I. J. (1987) Plant Mol Biol Reporter 5,346–349) is separated on 1% Tris-borate (TBE) agarose gels, transferredto nylon membranes (Amersham) and probed with ³²P-labeled random primedDNA sequences corresponding to a 0.7 kb BamHI/HindIII DNA fragment frompC8 containing a portion of the rps7/12 plastid targeting sequence.Homoplasmic shoots are rooted aseptically on spectinomycin-containingMS/IBA medium (McBride, K. E. et al. (1994) PNAS 91, 7301–7305) andtransferred to the greenhouse.

V. Breeding and Seed Production A. Breeding

The plants obtained via tranformation with a nucleic acid sequence ofthe present invention can be any of a wide variety of plant species,including those of monocots and dicots; however, the plants used in themethod of the invention are preferably selected from the list ofagronomically important target crops set forth supra. The expression ofa gene of the present invention in combination with othercharacteristics important for production and quality can be incorporatedinto plant lines through breeding. Breeding approaches and techniquesare known in the art. See, for example, Welsh J. R., Fundamentals ofPlant Genetics and Breeding, John Wiley & Sons, NY (1981); CropBreeding, Wood D. R. (Ed.) American Society of Agronomy Madison, Wis.(1983); Mayo O., The Theory of Plant Breeding, Second Edition, ClarendonPress, Oxford (1987); Singh, D. P., Breeding for Resistance to Diseasesand Insect Pests, Springer-Verlag, NY (1986); and Wricke and Weber,Quantitative Genetics and Selection Plant Breeding, Walter de Gruyterand Co., Berlin (1986).

The genetic properties engineered into the transgenic seeds and plantsdescribed above are passed on by sexual reproduction or vegetativegrowth and can thus be maintained and propagated in progeny plants.Generally said maintenance and propagation make use of knownagricultural methods developed to fit specific purposes such as tilling,sowing or harvesting. Specialized processes such as hydroponics orgreenhouse technologies can also be applied. As the growing crop isvulnerable to attack and damages caused by insects or infections as wellas to competition by weed plants, measures are undertaken to controlweeds, plant diseases, insects, nematodes, and other adverse conditionsto improve yield. These include mechanical measures such a tillage ofthe soil or removal of weeds and infected plants, as well as theapplication of agrochemicals such as herbicides, fungicides,gametocides, nematicides, growth regulants, ripening agents andinsecticides.

Use of the advantageous genetic properties of the transgenic plants andseeds according to the invention can further be made in plant breeding,which aims at the development of plants with improved properties such astolerance of pests, herbicides, or stress, improved nutritional value,increased yield, or improved structure causing less loss from lodging orshattering. The various breeding steps are characterized by well-definedhuman intervention such as selecting the lines to be crossed, directingpollination of the parental lines, or selecting appropriate progenyplants. Depending on the desired properties, different breeding measuresare taken. The relevant techniques are well known in the art and includebut are not limited to hybridization, inbreeding, backcross breeding,multiline breeding, variety blend, interspecific hybridization,aneuploid techniques, etc. Hybridization techniques also include thesterilization of plants to yield male or female sterile plants bymechanical, chemical, or biochemical means. Cross pollination of a malesterile plant with pollen of a different line assures that the genome ofthe male sterile but female fertile plant will uniformly obtainproperties of both parental lines. Thus, the transgenic seeds and plantsaccording to the invention can be used for the breeding of improvedplant lines, that for example, increase the effectiveness ofconventional methods such as herbicide or pesticide treatment or allowone to dispense with said methods due to their modified geneticproperties. Alternatively new crops with improved stress tolerance canbe obtained, which, due to their optimized genetic “equipment”, yieldharvested product of better quality than products that were not able totolerate comparable adverse developmental conditions.

B. Seed Production

In seed production, germination quality and uniformity of seeds areessential product characteristics. As it is difficult to keep a cropfree from other crop and weed seeds, to control seedborne diseases, andto produce seed with good germination, fairly extensive and well-definedseed production practices have been developed by seed producers, who areexperienced in the art of growing, conditioning and marketing of pureseed. Thus, it is common practice for the farmer to buy certified seedmeeting specific quality standards instead of using seed harvested fromhis own crop. Propagation material to be used as seeds is customarilytreated with a protectant coating comprising herbicides, insecticides,fungicides, bactericides, nematicides, molluscicides, or mixturesthereof. Customarily used protectant coatings comprise compounds such ascaptan, carboxin, thiram (TMTD®), methalaxyl (Apron®), andpirimiphos-methyl (Actellic®). If desired, these compounds areformulated together with further carriers, surfactants orapplication-promoting adjuvants customarily employed in the art offormulation to provide protection against damage caused by bacterial,fungal or animal pests. The protectant coatings may be applied byimpregnating propagation material with a liquid formulation or bycoating with a combined wet or dry formulation. Other methods ofapplication are also possible such as treatment directed at the buds orthe fruit.

VI. Alteration of Expression of Nucleic Acid Molecules

The alteration in expression of the nucleic acid molecules of thepresent invention is achieved in one of the following ways:

A. “Sense” Suppression

Alteration of the expression of a nucleotide sequence of the presentinvention, preferably reduction of its expression, is obtained by“sense” suppression (referenced in e.g. Jorgensen et al. (1996) PlantMol. Biol. 31, 957–973). In this case, the entirety or a portion of anucleotide sequence of the present invention is comprised in a DNAmolecule. The DNA molecule is preferably operatively linked to apromoter functional in a cell comprising the target gene, preferably aplant cell, and introduced into the cell, in which the nucleotidesequence is expressible. The nucleotide sequence is inserted in the DNAmolecule in the “sense orientation”, meaning that the coding strand ofthe nucleotide sequence can be transcribed. In a preferred embodiment,the nucleotide sequence is fully translatable and all the geneticinformation comprised in the nucleotide sequence, or portion thereof, istranslated into a polypeptide. In another preferred embodiment, thenucleotide sequence is partially translatable and a short peptide istranslated. In a preferred embodiment, this is achieved by inserting atleast one premature stop codon in the nucleotide sequence, which bringtranslation to a halt. In another more preferred embodiment, thenucleotide sequence is transcribed but no translation product is beingmade. This is usually achieved by removing the start codon, e.g. the“ATG”, of the polypeptide encoded by the nucleotide sequence. In afurther preferred embodiment, the DNA molecule comprising the nucleotidesequence, or a portion thereof, is stably integrated in the genome ofthe plant cell. In another preferred embodiment, the DNA moleculecomprising the nucleotide sequence, or a portion thereof, is comprisedin an extrachromosomally replicating molecule.

In transgenic plants containing one of the DNA molecules describedimmediately above, the expression of the nucleotide sequencecorresponding to the nucleotide sequence comprised in the DNA moleculeis preferably reduced. Preferably, the nucleotide sequence in the DNAmolecule is at least 70% identical to the nucleotide sequence theexpression of which is reduced, more preferably it is at least 80%identical, yet more preferably at least 90% identical, yet morepreferably at least 95% identical, yet more preferably at least 99%identical.

B. “Anti-sense” Suppression

In another preferred embodiment, the alteration of the expression of anucleotide sequence of the present invention, preferably the reductionof its expression is obtained by “anti-sense” suppression. The entiretyor a portion of a nucleotide sequence of the present invention iscomprised in a DNA molecule. The DNA molecule is preferably operativelylinked to a promoter functional in a plant cell, and introduced in aplant cell, in which the nucleotide sequence is expressible. Thenucleotide sequence is inserted in the DNA molecule in the “anti-senseorientation”, meaning that the reverse complement (also called sometimesnon-coding strand) of the nucleotide sequence can be transcribed. In apreferred embodiment, the DNA molecule comprising the nucleotidesequence, or a portion thereof, is stably integrated in the genome ofthe plant cell. In another preferred embodiment the DNA moleculecomprising the nucleotide sequence, or a portion thereof, is comprisedin an extrachromosomally replicating molecule. Several publicationsdescribing this approach are cited for further illustration (Green, P.J. et al., Ann. Rev. Biochem. 55:569–597 (1986); van der Krol, A. R. etal, Antisense Nuc. Acids & Proteins, pp. 125–141 (1991); Abel, P. P. etal., PNASroc. Natl. Acad. Sci. USA 86:6949–6952 (1989); Ecker, J. R. etal., Proc. Natl. Acad. Sci. USANAS 83:5372–5376 (August 1986)).

In transgenic plants containing one of the DNA molecules describedimmediately above, the expression of the nucleotide sequencecorresponding to the nucleotide sequence comprised in the DNA moleculeis preferably reduced. Preferably, the nucleotide sequence in the DNAmolecule is at least 70% identical to the nucleotide sequence theexpression of which is reduced, more preferably it is at least 80%identical, yet more preferably at least 90% identical, yet morepreferably at least 95% identical, yet more preferably at least 99%identical.

C. Homologous Recombination

In another preferred embodiment, at least one genomic copy correspondingto a nucleotide sequence of the present invention is modified in thegenome of the plant by homologous recombination as further illustratedin Paszkowski et al., EMBO Journal 7:4021–26 (1988). This technique usesthe property of homologous sequences to recognize each other and toexchange nucleotide sequences between each by a process known in the artas homologous recombination. Homologous recombination can occur betweenthe chromosomal copy of a nucleotide sequence in a cell and an incomingcopy of the nucleotide sequence introduced in the cell bytransformation. Specific modifications are thus accurately introduced inthe chromosomal copy of the nucleotide sequence. In one embodiment, theregulatory elements of the nucleotide sequence of the present inventionare modified. Such regulatory elements are easily obtainable byscreening a genomic library using the nucleotide sequence of the presentinvention, or a portion thereof, as a probe. The existing regulatoryelements are replaced by different regulatory elements, thus alteringexpression of the nucleotide sequence, or they are mutated or deleted,thus abolishing the expression of the nucleotide sequence. In anotherembodiment, the nucleotide sequence is modified by deletion of a part ofthe nucleotide sequence or the entire nucleotide sequence, or bymutation. Expression of a mutated polypeptide in a plant cell is alsocontemplated in the present invention. More recent refinements of thistechnique to disrupt endogenous plant genes have been described (Kempinet al., Nature 389:802–803 (1997) and Miao and Lam, Plant J., 7:359–365(1995).

In another preferred embodiment, a mutation in the chromosomal copy of anucleotide sequence is introduced by transforming a cell with a chimericoligonucleotide composed of a contiguous stretch of RNA and DNA residuesin a duplex conformation with double hairpin caps on the ends. Anadditional feature of the oligonucleotide is for example the presence of2′-O-methylation at the RNA residues. The RNA/DNA sequence is designedto align with the sequence of a chromosomal copy of a nucleotidesequence of the present invention and to contain the desired nucleotidechange. For example, this technique is further illustrated in U.S. Pat.No. 5,501,967 and Zhu et al. (1999) Proc. Natl. Acad. Sci. USA 96:8768–8773.

D. Ribozymes

In a further embodiment, the RNA coding for a polypeptide of the presentinvention is cleaved by a catalytic RNA, or ribozyme, specific for suchRNA. The ribozyme is expressed in transgenic plants and results inreduced amounts of RNA coding for the polypeptide of the presentinvention in plant cells, thus leading to reduced amounts of polypeptideaccumulated in the cells. This method is further illustrated in U.S.Pat. No. 4,987,071.

E. Dominant-Negative Mutants

In another preferred embodiment, the activity of the polypeptide encodedby the nucleotide sequences of this invention is changed. This isachieved by expression of dominant negative mutants of the proteins intransgenic plants, leading to the loss of activity of the endogenousprotein.

F. Aptamers

In a further embodiment, the activity of polypeptide of the presentinvention is inhibited by expressing in transgenic plants nucleic acidligands, so-called aptamers, which specifically bind to the protein.Aptamers are preferentially obtained by the SELEX (Systematic Evolutionof Ligands by EXponential Enrichment) method. In the SELEX method, acandidate mixture of single stranded nucleic acids having regions ofrandomized sequence is contacted with the protein and those nucleicacids having an increased affinity to the target are partitioned fromthe remainder of the candidate mixture. The partitioned nucleic acidsare amplified to yield a ligand enriched mixture. After severaliterations a nucleic acid with optimal affinity to the polypeptide isobtained and is used for expression in transgenic plants. This method isfurther illustrated in U.S. Pat. No. 5,270,163.G. Zinc Finger Proteins

A zinc finger protein that binds a nucleotide sequence of the presentinvention or to its regulatory region is also used to alter expressionof the nucleotide sequence. Preferably, transcription of the nucleotidesequence is reduced or increased. Zinc finger proteins are for exampledescribed in Beerli et al. (1998) PNAS PNAS 95:14628–14633., or in WO95/19431, WO 98/54311, or WO 96/06166, all incorporated herein byreference in their entirety.

H. dsRNA

Alteration of the expression of a nucleotide sequence of the presentinvention is also obtained by dsRNA interference as described forexample in WO 99/32619, WO 99/53050 or WO 99/61631, all incorporatedherein by reference in their entirety. In another preferred embodiment,the alteration of the expression of a nucleotide sequence of the presentinvention, preferably the reduction of its expression, is obtained bydouble-stranded RNA (dsRNA) interference. The entirety or, preferably aportion of a nucleotide sequence of the present invention is comprisedin a DNA molecule. The size of the DNA molecule is preferably from 100to 1000 nucleotides or more; the optimal size to be determinedempirically. Two copies of the identical DNA molecule are linked,separated by a spacer DNA molecule, such that the first and secondcopies are in opposite orientations. In the preferred embodiment, thefirst copy of the DNA molecule is in the reverse complement (also knownas the non-coding strand) and the second copy is the coding strand; inthe most preferred embodiment, the first copy is the coding strand, andthe second copy is the reverse complement. The size of the spacer DNAmolecule is preferably 200 to 10,000 nucleotides, more preferably 400 to5000 nucleotides and most preferably 600 to 1500 nucleotides in length.The spacer is preferably a random piece of DNA, more preferably a randompiece of DNA without homology to the target organism for dsRNAinterference, and most preferably a functional intron which iseffectively spliced by the target organism. The two copies of the DNAmolecule separated by the spacer are operatively linked to a promoterfunctional in a plant cell, and introduced in a plant cell, in which thenucleotide sequence is expressible. In a preferred embodiment, the DNAmolecule comprising the nucleotide sequence, or a portion thereof, isstably integrated in the genome of the plant cell. In another preferredembodiment the DNA molecule comprising the nucleotide sequence, or aportion thereof, is comprised in an extrachromosomally replicatingmolecule. Several publications describing this approach are cited forfurther illustration (Waterhouse et al. (1998) PNAS 95:13959–13964;Chuang and Meyerowitz (2000) PNAS 97:4985–4990; Smith et al. (2000)Nature 407:319–320). Alteration of the expression of a nucleotidesequence by dsRNA interference is also described in, for example WO99/32619, WO 99/53050 or WO 99/61631, all incorporated herein byreference in their entirety

In transgenic plants containing one of the DNA molecules describedimmediately above, the expression of the nucleotide sequencecorresponding to the nucleotide sequence comprised in the DNA moleculeis preferably reduced. Preferably, the nucleotide sequence in the DNAmolecule is at least 70% identical to the nucleotide sequence theexpression of which is reduced, more preferably it is at least 80%identical, yet more preferably at least 90% identical, yet morepreferably at least 95% identical, yet more preferably at least 99%identical.

I. Insertion of a DNA Molecule (Insertional Mutagenesis)

In another preferred embodiment, a DNA molecule is inserted into achromosomal copy of a nucleotide sequence of the present invention, orinto a regulatory region thereof. Preferably, such DNA moleculecomprises a transposable element capable of transposition in a plantcell, such as e.g. Ac/Ds, Em/Spm, mutator. Alternatively, the DNAmolecule comprises a T-DNA border of an Agrobacterium T-DNA. The DNAmolecule may also comprise a recombinase or integrase recognition sitewhich can be used to remove part of the DNA molecule from the chromosomeof the plant cell. Methods of insertional mutagenesis using T-DNA,transposons, oligonucleotides or other methods known to those skilled inthe art are also encompassed. Methods of using T-DNA and transposon forinsertional mutagenesis are described in Winkler et al. (1989) MethodsMol. Biol. 82:129–136 and Martienssen (1998) PNAS 95:2021–2026,incorporated herein by reference in their entireties.

J. Deletion Mutagenesis

In yet another embodiment, a mutation of a nucleic acid molecule of thepresent invention is created in the genomic copy of the sequence in thecell or plant by deletion of a portion of the nucleotide sequence orregulator sequence. Methods of deletion mutagenesis are known to thoseskilled in the art. See, for example, Miao et al, (1995) Plant J. 7:359.

In yet another embodiment, this deletion is created at random in a largepopulation of plants by chemical mutagenesis or irradiation and a plantwith a deletion in a gene of the present invention is isolated byforward or reverse genetics. Irradiation with fast neutrons or gammarays is known to cause deletion mutations in plants (Silverstone et al,(1998) Plant Cell, 10:155–169; Bruggemann et al., (1996) Plant J.,10:755–760; Redei and Koncz in Methods in Arabidopsis Research, WorldScientific Press (1992), pp. 16–82). Deletion mutations in a gene of thepresent invention can be recovered in a reverse genetics strategy usingPCR with pooled sets of genomic DNAs as has been shown in C. elegans(Liu et al., (1999), Genome Research, 9:859–867.). A forward geneticsstrategy would involve mutagenesis of a line displaying PTGS followed byscreening the M2 progeny for the absence of PTGS. Among these mutantswould be expected to be some that disrupt a gene of the presentinvention. This could be assessed by Southern blot or PCR for a gene ofthe present invention with genomic DNA from these mutants.

K. Overexpression in a Plant Cell

In yet another preferred embodiment, a nucleotide sequence of thepresent invention encoding a polypeptide is over-expressed. Examples ofnucleic acid molecules and expression cassettes for over-expression of anucleic acid molecule of the present invention are described above.Methods known to those skilled in the art of over-expression of nucleicacid molecules are also encompassed by the present invention.

In a preferred embodiment, the expression of the nucleotide sequence ofthe present invention is altered in every cell of a plant. This is forexample obtained though homologous recombination or by insertion in thechromosome. This is also for example obtained by expressing a sense orantisense RNA, zinc finger protein or ribozyme under the control of apromoter capable of expressing the sense or antisense RNA, zinc fingerprotein or ribozyme in every cell of a plant. Constitutive expression,inducible, tissue-specific or developmentally regulated expression arealso within the scope of the present invention and result in aconstitutive, inducible, tissue-specific or developmentally-regulatedalteration of the expression of a nucleotide sequence of the presentinvention in the plant cell. Constructs for expression of the sense orantisense RNA, zinc finger protein or ribozyme, or for over-expressionof a nucleotide sequence of the present invention, are prepared andtransformed into a plant cell according to the teachings of the presentinvention, e.g. as described infra.

VII. Polypeptides

The present invention further relates to isolated polypeptidescomprising the amino acid sequence of SEQ ID NO:2. In particular,isolated polypeptides comprising the amino acid sequence of SEQ ID NO:2,and variants having conservative amino acid modifications. One skilledin the art will recognize that individual substitutions, deletions oradditions to a nucleic acid, peptide, polypeptide or protein sequencewhich alters, adds or deletes a single amino acid or a small percent ofamino acids in the encoded sequence is a “conservative modification”where the modification results in the substitution of an amino acid witha chemically similar amino acid. Conservative modified variants providesimilar biological activity as the unmodified polypeptide. Conservativesubstitution tables listing functionally similar amino acids are knownin the art. See Crighton (1984) Proteins, W. H. Freeman and Company.

In a preferred embodiment, a polypeptide having substantial similarityto a polypeptide sequence listed in SEQ ID NO:2, or exon, domain, orfeature thereof, is an allelic variant of the polypeptide sequencelisted in red SEQ ID NO:2. In another preferred embodiment, apolypeptide having substantial similarity to a polypeptide sequencelisted in SEQ ID NO:2, or exon, domain, or feature thereof, is anaturally occurring variant of the polypeptide sequence listed in SEQ IDNO:2. In another preferred embodiment, a polypeptide having substantialsimilarity to a polypeptide sequence listed in SEQ ID NO:2, or exon,domain, or feature thereof, is a polymorphic variant of the polypeptidesequence listed in SEQ ID NO:2.

In an alternate preferred embodiment, the sequence having substantialsimilarity contains a deletion or insertion of at least one amino acid.In a more preferred embodiment, the deletion or insertion is of lessthan about ten amino acids. In a most preferred embodiment, the deletionor insertion is of less than about three amino acids.

In a preferred embodiment, the sequence having substantial similarityencodes a substitution in at least one amino acid.

Embodiments of the present invention also contemplate an isolatedpolypeptide containing a polypeptide sequence including:

-   -   (f) a polypeptide sequence listed in SEQ ID NO:2, or exon,        domain, or feature thereof;    -   (g) a polypeptide sequence having substantial similarity to (a);    -   (h) a polypeptide sequence encoded by a nucleotide sequence        identical to or having substantial similarity to a nucleotide        sequence listed in SEQ ID NO:1, or an exon, domain, or feature        thereof, or a sequence complementary thereto;    -   (i) a polypeptide sequence encoded by a nucleotide sequence        capable of hybridizing under medium stringency conditions to a        nucleotide sequence listed in SEQ ID NO:1, or to a sequence        complementary thereto; and    -   (j) a functional fragment of (a), (b), (c) or (d).

In another preferred embodiment, the polypeptide having substantialsimilarity is an allelic variant of a polypeptide sequence listed in SEQID NO:2, or a fragment, domain, repeat, feature, or chimeras thereof. Inanother preferred embodiment, the isolated nucleic acid includes aplurality of regions from the polypeptide sequence encoded by anucleotide sequence identical to or having substantial similarity to anucleotide sequence listed in SEQ ID NO:1, or fragment, domain, orfeature thereof, or a sequence complementary thereto.

In another preferred embodiment, the polypeptide is a polypeptidesequence listed in SEQ ID NO:2. In another preferred embodiment, thepolypeptide is a functional fragment or domain. In yet another preferredembodiment, the polypeptide is a chimera, where the chimera may includefunctional protein domains, including domains, repeats,post-translational modification sites, or other features. In a morepreferred embodiment, the polypeptide is a plant polypeptide. In a morepreferred embodiment, the plant is a dicot. In a more preferredembodiment, the plant is a gymnosperm. In a more preferred embodiment,the plant is a monocot. In a more preferred embodiment, the monocot is acereal. In a more preferred embodiment, the cereal may be, for example,maize, wheat, barley, oats, rye, millet, sorghum, triticale, secale,einkorn, spelt, emmer, teff, milo, flax, gramma grass, Tripsacum, andteosinte. In a most preferred embodiment, the cereal is rice.

In a preferred embodiment, the polypeptide is expressed in a specificlocation or tissue of a plant. In a more preferred embodiment, thelocation or tissue is for example, but not limited to, epidermis,vascular tissue, meristem, cambium, cortex or pith. In a most preferredembodiment, the location or tissue is leaf or sheath, root, flower, anddeveloping ovule or seed. In a more preferred embodiment, the locationor tissue may be, for example, epidermis, root, vascular tissue,meristem, cambium, cortex, pith, leaf, and flower. In a more preferredembodiment, the location or tissue is a seed.

In a preferred embodiment, the polypeptide sequence encoded by anucleotide sequence having substantial similarity to a nucleotidesequence listed in SEQ ID NO:1 or a fragment, domain, or feature thereofor a sequence complementary thereto, includes a deletion or insertion ofat least one nucleotide. In a more preferred embodiment, the deletion orinsertion is of less than about thirty nucleotides. In a most preferredembodiment, the deletion or insertion is of less than about fivenucleotides.

In a preferred embodiment, the polypeptide sequence encoded by anucleotide sequence having substantial similarity to a nucleotidesequence listed in SEQ ID NO:1, or fragment, domain, or feature thereofor a sequence complementary thereto, includes a substitution of at leastone codon. In a more preferred embodiment, the substitution isconservative.

In a preferred embodiment, the polypeptide sequences having substantialsimilarity to the polypeptide sequence listed in SEQ ID NO:2, or afragment, domain, repeat, feature, or chimeras thereof includes adeletion or insertion of at least one amino acid.

The polypeptides of the invention, fragments thereof or variants thereofcan comprise any number of contiguous amino acid residues from apolypeptide of the invention, wherein the number of residues is selectedfrom the group of integers consisting of from 10 to the number ofresidues in a full-length polypeptide of the invention. Preferably, theportion or fragment of the polypeptide is a functional protein. Thepresent invention includes active polypeptides having specific activityof at least 20%, 30%, or 40%, and preferably at least 505, 60%, or 70%,and most preferably at least 805, 90% or 95% that of the native(non-synthetic) endogenous polypeptide. Further, the substratespecificity (k_(cat)/K_(m)) is optionally substantially similar to thenative (non-synthetic), endogenous polypeptide. Typically the K_(m) willbe at least 30%, 40%, or 50% of the native, endogenous polypeptide; andmore preferably at least 605, 70%, 80%, or 90%. Methods of assaying andquantifying measures of activity and substrate specificity are wellknown to those of skill in the art.

The isolated polypeptides of the present invention will elicitproduction of an antibody specifically reactive to a polypeptide of thepresent invention when presented as an immunogen. Therefore, thepolypeptides of the present invention can be employed as immunogens forconstructing antibodies immunoreactive to a protein of the presentinvention for such purposes, but not limited to, immunoassays or proteinpurification techniques. Immunoassays for determining binding are wellknown to those of skill in the art such as, but not limited to, ELISAsor competitive immunoassays.

Embodiments of the present invention also relate to chimericpolypeptides encoded by the isolated nucleic acid molecules of thepresent disclosure including a chimeric polypeptide containing apolypeptide sequence encoded by an isolated nucleic acid containing anucleotide sequence including:

-   -   (g) a nucleotide sequence listed in SEQ ID NO:1, or exon,        domain, or feature thereof;    -   (h) a nucleotide sequence having substantial similarity to (a);    -   (i) a nucleotide sequence capable of hybridizing to (a);    -   (j) a nucleotide sequence complementary to (a), (b) or (c); and    -   (k) a nucleotide sequence which is the reverse complement of        (a), (b) or (c);    -   (l) or a functional fragment thereof.

A polypeptide containing a polypeptide sequence encoded by an isolatednucleic acid containing a nucleotide sequence, its complement, or itsreverse complement, encoding a polypeptide including a polypeptidesequence including:

-   -   (g) a polypeptide sequence listed in SEQ ID NO:2, or a domain,        repeat, feature, or chimeras thereof;    -   (h) a polypeptide sequence having substantial similarity to (a);    -   (i) a polypeptide sequence encoded by a nucleotide sequence        identical to or having substantial similarity to a nucleotide        sequence listed in SEQ ID NO:1, or an exon, domain, or feature        thereof, or a sequence complementary thereto;    -   (j) a polypeptide sequence encoded by a nucleotide sequence        capable of hybridizing under medium stringency conditions to a        nucleotide sequence listed in SEQ ID NO:1, or to a sequence        complementary thereto; and    -   (k) a functional fragment of (a), (b), (c) or (d);    -   (l) or a functional fragment thereof.

The isolated nucleic acid molecules of the present invention are usefulfor expressing a polypeptide of the present invention in a recombinantlyengineered cell such as a bacteria, yeast, insect, mammalian or plantcell. The cells produce the polypeptide in a non-natural condition (e.g.in quantity, composition, location and/or time) because they have beengenetically altered to do so. Those skilled in the art are knowledgeablein the numerous expression systems available for expression of nucleicacids encoding a protein of the present invention, and will not bedescribed in detail below.

Briefly, the expression of isolated nucleic acids encoding a polypeptideof the invention will typically be achieved, for example, by operablylinking the nucleic acid or cDNA to a promoter (constitutive orregulatable) followed by incorporation into an expression vector. Thevectors are suitable for replication and/or integration in eitherprokaryotes or eukaryotes. Commonly used expression vectors comprisetranscription and translation terminators, initiation sequences andpromoters for regulation of the expression of the nucleic acid moleculeencoding the polypeptide. To obtain high levels of expression of thecloned nucleic acid molecule, it is desirable to use expression vectorscomprising a strong promoter to direct transcription, a ribosome-bindingsite for translation initiation, and a transcription/translationterminator. One skilled in the art will recognize that modifications maybe made to the polypeptide of the present invention without diminishingits biological activity. Some modifications may be made to facilitatethe cloning, expression or incorporation of the polypeptide of theinvention into a fusion protein. Such modification are well known in theart and include, but are not limited to, a methionine added at the aminoterminus to provide an initiation site, or additiona amino acids (e.g.poly Histadine) placed on either terminus to create conveniently locatedpurification sequences. Restriction sites or termination codons can alsobe introduced into the vector.

In a preferred embodiment, the expression vector includes one or moreelements such as, for example, but not limited to, a promoter-enhancersequence, a selection marker sequence, an origin of replication, anepitope-tag encoding sequence, or an affinity purification-tag encodingsequence. In a more preferred embodiment, the promoter-enhancer sequencemay be, for example, the CaMV 35S promoter, the CaMV 19S promoter, thetobacco PR-1a promoter, the ubiquitin promoter, and the phaseolinpromoter. In another embodiment, the promoter is operable in plants, andmore preferably, a constitutive or inducible promoter. In anotherpreferred embodiment, the selection marker sequence encodes anantibiotic resistance gene. In another preferred embodiment, theepitope-tag sequence encodes V5, the peptide Phe-His-His-Thr-Thr,hemagglutinin, or glutathione-S-transferase. In another preferredembodiment the affinity purification-tag sequence encodes a polyaminoacid sequence or a polypeptide. In a more preferred embodiment, thepolyamino acid sequence is polyhistidine. In a more preferredembodiment, the polypeptide is chitin binding domain orglutathione-S-transferase. In a more preferred embodiment, the affinitypurification-tag sequence comprises an intein encoding sequence.

Prokaryotic cells may be used a host cells, for example, but not limitedto, Escherichia coli, and other microbial strains known to those in theart. Methods for expressing proteins in prokaryotic cells are well knownto those in the art and can be found in many laboratory manuals such asMolecular Cloning: A Laboratory Manual, by J. Sambrook et al. (1989,Cold Spring Harbor Laboratory Press). A variety of promoters, ribosomebinding sites, and operators to control expression are available tothose skilled in the art, as are selectable markers such as antibioticresistance genes. The type of vector chosen is to allow for optimalgrowth and expression in the selected cell type.

A variety of eukaryotic expression systems are available such as, butnot limited to, yeast, insect cell lines, plant cells and mammaliancells. Expression and synthesis of heterologous proteins in yeast iswell known (see Sherman et al., Methods in Yeast Genetics, Cold SpringHarbor Laboratory Press, 1982). Commonly used yeast strains widely usedfor production of eukaryotic proteins are Saccharomyces cerevisiae andPichia pastoris, and vectors, strains and protocols for expression areavailable from commercial suppliers (e.g., Invitrogen).

Mammalian cell systems may be transfected with expression vectors forproduction of proteins. Many suitable host cell lines are available tothose in the art, such as, but not limited to the HEK293, BHK21 and CHOcells lines. Expression vectors for these cells can include expressioncontrol sequences such as an origin of replication, a promoter, (e.g.,the CMV promoter, a HSV tk promoter or phosphoglycerate kinase (pgk)promoter), an enhancer, and protein processing sites such as ribosomebinding sites, RNA splice sites, polyadenylation sites, andtranscription terminator sequences. Other animal cell lines useful forthe production of proteins are available commercially or fromdepositories such as the American Type Culture Collection.

Expression vectors for expressing proteins in insect cells are usuallyderived from the SF9 baculovirus or other viruses known in the art. Anumber of suitable insect cell lines are available including but notlimited to, mosquito larvae, silkworm, armyworm, moth and Drosophilacell lines.

Methods of transfecting animal and lower eukaryotic cells are known.Numerous methods are used to make eukaryotic cells competent tointroduce DNA such as but not limited to: calcium phosphateprecipitation, fusion of the recipient cell with bacterial protoplastscontaining the DNA, treatment of the recipient cells with liposomescontaining the DNA, DEAE dextrin, electroporation, biolistics, andmicroinjection of the DNA directly into the cells. Tranfected cells arecultured using means well known in the art (see, Kuchler, R. J.,Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson andRoss, Inc. 1997).

Once a polypeptide of the present invention is expressed it may beisolated and purified from the cells using methods known to thoseskilled in the art. The purification process may be monitored usingWestern blot techniques or radioimmunoassay or other standardimmunoassay techniques. Protein purification techniques are commonlyknown and used by those in the art (see R. Scopes, Protein Purification:Principles and Practice, Springer-Verlag, New York 1982: Deutscher,Guide to Protein Purification, Academic Press (1990). Embodiments of thepresent invention provide a method of producing a recombinant protein inwhich the expression vector includes one or more elements including apromoter-enhancer sequence, a selection marker sequence, an origin ofreplication, an epitope-tag encoding sequence, and an affinitypurification-tag encoding sequence. In one preferred embodiment, thenucleic acid construct includes an epitope-tag encoding sequence and theisolating step includes use of an antibody specific for the epitope-tag.In another preferred embodiment, the nucleic acid construct contains apolyamino acid encoding sequence and the isolating step includes use ofa resin comprising a polyamino acid binding substance, preferably wherethe polyamino acid is polyhistidine and the polyamino binding resin isnickel-charged agarose resin. In yet another preferred embodiment, thenucleic acid construct contains a polypeptide encoding sequence and theisolating step includes the use of a resin containing a polypeptidebinding substance, preferably where the polypeptide is a chitin bindingdomain and the resin contains chitin-sepharose.

The polypeptides of the present invention cam be synthesized usingnon-cellular synthetic methods known to those in the art. Techniques forsolid phase synthesis are described by Barany and Mayfield, Solid-PhasePeptide Synthesis, pp. 3–284 in the Peptides: Analysis, Synthesis,Biology, Vol. 2, Special Methods in Peptide Synthesis, Part A;Merrifield, et al., J. Am. Chem. Soc. 85:2149–56 (1963) and Stewart etal., Solid Phase Peptide Synthesis, 2^(nd) ed. Pierce Chem. Co.,Rockford, Ill. (1984).

The present invention further provides a method for modifying (i.e.increasing or decreasing) the concentration or composition of thepolypeptides of the invention in a plant or part thereof. Modificationcan be effected by increasing or decreasing the concentration and/or thecomposition (i.e. the ration of the polypeptides of the presentinvention) in a plant. The method comprised introducing into a plantcell with an expression cassette comprising a nucleic acid molecule ofthe present invention, or an nucleic acid encoding a RAR1 sequence asdescribed above to obtain a transformed plant cell or tissue, culturingthe transformed plant cell or tissue. The nucleic acid molecule can beunder the regulation of a constitutive or inducible promoter. The methodcan further comprise inducing or repressing expression of a nucleic acidmolecule of a sequence in the plant for a time sufficient to modify theconcentration and/or composition in the plant or plant part.

A plant or plant part having modified expression of a nucleic acidmolecule of the invention can be analyzed and selected using methodsknown to those skilled in the an such as, but not limited to, Southernblot, DNA sequencing, or PCR analysis using primers specific to thenucleic acid molecule and detecting amplicons produced therefrom.

In general, concentration or composition in increased or decreased by atleast 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% relative to anative control plant, plant part or cell lacking the expressioncassette.

VIII. BOS1 Promoter

In addition, the present invention also provides promoters capable ofconferring abiotic stress responsive expression to an associatednucleotide sequence of interest. Preferred are promoter sequencesobtainable from the Arabidopsis thaliana BOS1 gene. Nucleotide sequencescomprising functional and/or structural equivalents thereof are alsoembraced by the invention. The present invention thus relates tonucleotide sequences that function as promoters of transcription ofassociated nucleotide sequences. The promoter region may also includeelements that act as regulators of gene expression such as activators,enhancers, and/or repressors and may include the 5′ non-translatedleader sequence of the transcribed mRNA and/or introns and, optionally,exons.

Abiotic stress responsive inducible expression means that the nucleotidesequence of interest is preferentially expressed when an abiotic stressaccording to the invention is applied. Thus, the nucleotide sequenceaccording to the invention is useful for abiotic stress responsiveinducible expression of an associated nucleotide sequence of interest,which preferably is a coding sequence. It is known to the skilledartisan that the associated coding sequence of interest can be expressedin sense or in antisense orientation. Further, the coding sequence ofinterest may be of heterologous or homologous origin with respect to theplant to be transformed. In case of a homologous coding sequence, thenucleotide sequence according to the invention is useful for ectopicexpression of said sequence. In one particular embodiment of theinvention expression of the coding sequence of interest under control ofa nucleotide sequence according to the invention suppresses its ownexpression and that of the original copy of the gene by a process calledco-suppression.

The promoters of the present invention can be obtained, for example,from Arabidopsis thaliana genomic DNA by probing an Arabidopsis genomiclibrary with a cDNA according to the invention using methods known inthe art. It is obvious to a person skilled in the art that genomic DNAfrom any other organism, particularly from plants, can be used to obtaina lipoxygenase promoter from any organism of interest. This genomic DNAis then sequenced and aligned to the cDNA sequence. Basically, allnucleotide sequences upstream of the start codon are considered to bepart of the lipoxygenase promoter region. In addition, introns and,optionally, exons can be added to this region to form a functionalpromoter that confers abiotic stress responsvie inducible expression toan associated coding region.

In a preferred embodiment of the invention, the BOS1 promoter is acomponent of SEQ ID NO:4. Other preferred embodiments of the inventionare the nucleotide sequences depicted in SEQ ID NOs:4 or 3.

Based on the sequence information given in SEQ ID NO:4 or 3, the DNAsequences of the invention can be obtained, for example, by PCR using anucleic acid molecule of SEQ ID NO:4 or genomic DNA from a plant or anyother organism of interest as template. The person skilled in the artknows how to arrive at such sequences using methods known in the art.These sequences then can be fused to reporter genes to demonstratepromoter activity. For example, chimeric genes can be constructed thatinclude part of the 5′ regulatory sequence of the BOS1 gene fused to theGFP coding sequence. To this end, SEQ ID NO:3 can be used as templatefor the polymerase chain reaction (PCR). Gene-specific primers can bedesigned to amplify the 5′ promoter region of the gene. Usingcombinations of, for example, the BOS1 promoter reverse primer (SEQ IDNO:14) with BOS1 promoter forward primer (SEQ ID NO:13) the regulatorysequences that are ˜1.2 kb and ˜2 kb upstream of the initiatingmethionine are isolated. The nucleotide sequence of the PCR fragmentamplified with BOS1 promoter forward primer and BOS1 promoter reverseprimer is shown in SEQ ID NO:4. For ease of cloning the primers consist,for example, of gene specific sequences and attB recombination sites forthe GATEWAY™ cloning technology (Life Technologies, GIBCO BRL,Rockville, Md. USA).

Transgenic plants are then produced using, for example,Agrobacterium-mediated transformation techniques. Expression of the genefusion protein can be monitored in transformants by confocal imagingusing a Leica-TCS confocal laser scanning microscope and a PLAPO ×100oil immersion objective (Leica Microsystems, Heidelberg, Germany) withthe following filter settings: excitation 476/488 nm; GFP-emission515–552 nm, chlorophyll-emission 673–695 nm. GFP fluorescence andchlorophyll autofluorescence are recorded simultaneously usingindependent 2-channel-detection. Confocal imaging of leaves fromtransgenic rice plants expressing the pRCI promoter::GFP construct canbe carried out to assay promoter activity in response to abiotic andbiotic inducers.

It is apparent to the skilled artisan that, based on the nucleotidesequences shown in SEQ ID NO:4 or 3, any primer combination of interestcan be chosen to PCR amplify DNA fragments of various lengths that canbe used according to the invention. Thus, any region of interest can beamplified from SEQ ID NOs:4 or 3. For example, primers can be designedto specifically amplify intron 1 or intron 2 or the 5′ upstream region.The 5′ upstream region is defined herein as the region between theputative TATA box and the putative start codon of the lipoxygenaseprotein.

Further, it might also be desirable to combine any of these sequenceswith the 3′ untranslated region of the lipoxygenase cDNA sequence (ofSEQ ID NO:5).

The invention thus includes fragments derived from the rice RCI-1lipoxygenase gene that function according to the invention, i.e. arecapable of conferring chemically induced but not wound- or pathogeninduced expression of an associated nucleotide sequence. This can betested by generating such promoter fragments, fusing them to aselectable or screenable marker gene and assaying the fusion constructsfor retention of promoter activity in transient expression assays withprotoplasts or in stably transformed plants. Such assays are within theskill of the ordinary artisan. Preferred nucleic acid molecule fragmentsof the invention are of at least about 500 bases, particularly ofbetween about 1000 bases and about 1500 bases, more particularly ofabout 2000 bases and most particularly of between about 3000 bases andabout 4500 bases in length.

It is also clear to the skilled artisan that mutations, insertions,deletions end/or substitutions of one or more nucleotides can beintroduced into the nucleotide sequences of SEQ ID NOs:3 or 4 or longeror shorter fragments derived from the sequence information thereof usingmethods known in the art. In addition, an unmodified or modifiednucleotide sequence of the present invention can be varied by shufflingthe sequence of the invention. To test for a function of variantnucleotide sequences according to the invention, the sequence ofinterest is operably linked to a selectable or screenable marker geneand expression of the marker gene is tested in transient expressionassays with protoplasts or in stably transformed plants. It is known tothe skilled artisan that nucleotide sequences capable of drivingexpression of an associated nucleotide sequence are build in a modularway. Accordingly, expression levels from shorter nucleic acid moleculefragments may be different than the one from the longest fragment andmay be different from each other. For example, deletion of adown-regulating upstream element will lead to an increase in theexpression levels of the associated nucleotide sequence while deletionof an up-regulating element will decrease the expression levels of theassociated nucleotide sequence.

Another way of identifying promoter elements necessary for regulatedexpression of an associated nucleotide sequence is the so-calledlinker-scanning analysis. Linker-scanning mutagenesis allows for theidentification of short defined motifs whose mutation alters thepromoter activity. Accordingly, a set of linker-scanning mutantpromoters fused to the coding sequence of the GUS reporter gene oranother marker gene can be constructed using methods known in the art.These construct are then transformed into Arabidopsis, for example, andGUS activity is assayed in several independent transgenic lines. Theeffect of each mutation on promoter activity is then compared to anequivalent number of transgenic lines containing an unmutated ricelipoxygenase gene promoter. It is expected, that when a motif is mutatedthat is involved in chemically, but not wound or pathogen-inducibleexpression, that the level of expression of the reporter gene ismodified. If, for example, a higher average induction of GUS activity bya chemical inducer is detected than the one from the control constructmost likely a negative regulatory element had been mutated in thisconstruct. If, on the other hand, a complete loss of inducibility of GUSactivity by a chemical regulator according to the invention is observed,most likely a positive regulatory element necessary chemical inductionhas been mutated. In a next step, particularly in the case of theputative positive regulatory element, the wild-type sequencescorresponding to the mutated fragments are fused to a minimal promoterand the newly created promoter is tested for the ability to conferregulated expression to an associated marker gene.

Embraced by the present invention are also functional equivalents of theRCI-1 promoters of the present invention, i.e. nucleotide sequences thathybridize under stringent conditions to any one of SEQ ID NO:3 or 4. Astringent hybridization is performed at a temperature of 65° C.,preferably 60° C. and most preferably 55° C. in double strength (2×)citrate buffered saline (SSC) containing 0.1% SDS followed by rinsing ofthe support at the same temperature but with a buffer having a reducedSSC concentration. Such reduced concentration buffers are typicallyone-tenth strength SSC (0.1×SSC) containing 0.1% SDS, preferably 0.2×SSCcontaining 0.1% SSC and most preferably half strength SSC (0.5×SSC)containing 0.1% SDS. In fact, functional equivalents to all or part ofthe RCI-1 lipoxygenase promoter from other organisms can be found byhybridizing any one of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 or the4.5 PstI/PstI fragment of plasmid pBSK+LOX4A which has been depositedunder accession no DSM 13524 with genomic DNA isolated from an organismof interest, particularly from another monocot. The skilled artisanknows how to proceed to find such sequences as there are many ways knownin the art to identify homologous sequences from other organisms. Suchnewly identified DNA molecules then can be sequenced and the sequencecan be compared to any one of SEQ ID NO:3 or 4, and tested for promoteractivity. Within the scope of the present invention are DNA moleculeshaving at least 75%, preferably 80%, more preferably 90%, and mostpreferably 95% sequence identity to the nucleotide sequence of any oneof SEQ ID NOs:3 or 4 over a length of at least 50 nucleotides. Thepercentage of sequence identity is determined using computer programsthat are based on dynamic programming algorithms. Computer programs thatare preferred within the scope of the present invention include theBLAST (Basic Local Alignment Search Tool) search programs designed toexplore all of the available sequence databases regardless of whetherthe query is protein or DNA. Version BLAST 2.0 (Gapped BLAST) of thissearch tool has been made publicly available on the Internet. It uses aheuristic algorithm that seeks local as opposed to global alignments andis therefore able to detect relationships among sequences which shareonly isolated regions. The scores assigned in a BLAST search have awell-defined statistical interpretation. Said programs are preferablyrun with optional parameters set to the default values.

If desired, the promoters of the present invention can be fused with thenucleotide sequence encoding a transit peptide according to theinvention for example, by using the nucleotide sequence depicted in SEQID NO:4, for abiotic stress responsive expression of an associatedcoding region of interest in plastids, particularly in chloroplasts.

It is another object of the present invention to provide recombinantnucleic acid molecules comprising a promoter according to the inventionoperably linked to a nucleotide sequence of interest. The nucleotidesequence of interest can, for example, code for a ribosomal RNA, anantisense RNA or any other type of RNA that is not translated intoprotein. In another preferred embodiment of the invention the nucleotidesequence of interest is translated into a protein product. Thenucleotide sequence associated with the promoter sequence may be ofhomologous or heterologous origin with respect to the plant to betransformed. The sequence may also be entirely or partially synthetic.Regardless of the origin, the associated nucleotide sequence will beexpressed in the transformed plant in accordance with the expressionproperties of the promoter to which it is linked. In case of homologousnucleotide sequences associated with the promoter sequence, the promoteraccording to the invention is useful for ectopic expression of saidhomologous sequences. Ectopic expression means that the nucleotidesequence associated with the promoter sequence is expressed in tissuesand organs and/or at times where said sequence may not be expressedunder control of its own promoter. In one particular embodiment of theinvention, expression of nucleotide sequence associated with thepromoter sequence suppresses its own expression and that of the originalcopy of the gene by a process called cosuppression.

In a preferred embodiment of the invention the associated nucleotidesequence may code for a protein that is desired to be expressed in aaabiotic stress responsive inducible fashion. Such nucleotide sequencespreferably encode proteins conferring a desirable phenotypic trait tothe plant transformed therewith. Examples are nucleotide sequencesencoding proteins conferring abiotic stress tolerance, antibioticresistance, virus resistance, insect resistance, disease resistance, orresistance to other pests, herbicide tolerance, improved nutritionalvalue, improved performance in an industrial process or alteredreproductive capability. The associated nucleotide sequence may also beone that is transferred to plants for the production of commerciallyvaluable enzymes or metabolites in the plant. Embraced by the presentinvention are also selectable or screenable marker genes, i.e. genescomprising a nucleotide sequence of the invention operably linked to acoding region encoding a selectable or screenable trait.

Examples of selectable or screenable marker genes are described below.For certain target species, different antibiotic or herbicide selectionmarkers may be preferred. Selection markers used routinely intransformation include the nptlI gene that confers resistance tokanamycin, paromomycin, geneticin and related antibiotics (Vieira andMessing, 1982, Gene 19: 259–268; Bevan et al., 1983, Nature 304:184–187)the bacterial aadA gene (Goldschmidt-Clermont, 1991, Nucl. Acids Res.19: 4083–4089), encoding aminoglycoside 3′-adenylyltransferase andconferring resistance to streptomycin or spectinomycin, the hph genewhich confers resistance to the antibiotic hygromycin (Blochlinger andDiggelmann, 1984, Mol. Cell. Biol. 4: 2929–2931), and the dhfr gene,which confers resistance to methotrexate (Bourouis and Jarry, 1983, EMBOJ. 2: 1099–1104). Other markers to be used include a phosphinothricinacetyltransferase gene, which confers resistance to the herbicidephosphinothricin (White et al., 1990, Nucl. Acids Res. 18: 1062; Spenceret al. 1990, Theor. Appl. Genet. 79: 625–631), a mutant EPSP synthasegene encoding glyphosate resistance (Hinchee et al., 1988,Bio/Technology 6: 915–922), a mutant acetolactate synthase (ALS) genewhich confers imidazolione or sulfonylurea resistance (Lee et al., 1988,EMBO J. 7: 1241–1248), a mutant psbA gene conferring resistance toatrazine (Smeda et al., 1993, Plant Physiol. 103: 911–917), or a mutantprotoporphyrinogen oxidase gene as described in EP 0 769 059. Selectionmarkers resulting in positive selection, such as a phosphomannoseisomerase gene, as described in patent application WO 93/05163, are alsoused.

Identification of transformed cells may also be accomplished throughexpression of screenable marker genes such as genes coding forchloramphenicol acetyl transferase (CAT), β-glucuronidase (GUS),luciferase (LUC), and green fluorescent protein (GFP) or any otherprotein that confers a phenotypically distinct trait to the transformedcell.

It is a further objective of the invention to provide recombinantexpression vectors comprising a nucleotide sequence of the inventionfused to an associated nucleotide sequence of interest. In thesevectors, foreign nucleic acid molecules can be inserted into apolylinker region such that these exogenous sequences can be expressedin a suited host cell which may be, for example, of bacterial or plantorigin. For example, the plasmid pBI101 derived from the Agrobacteriumtumefaciens binary vector pBIN19 allows cloning and testing of promotersusing beta-glucuronidase (GUS) expression signal (Jefferson et al, 1987,EMBO J 6: 3901–3907). The size of the vector is 12.2 kb. It has alow-copy RK2 origin of replication and confers kanamycine resistance inboth bacteria and plants. There are numerous other expression vectorsknown to the person skilled in the art that can be used according to theinvention.

The present invention also provides transgenic plants comprising therecombinant DNA sequences of the invention. The invention thus relatesto plant cells, to plants derived from such cells, to plant material, tothe progeny and to seeds derived from such plants, and to agriculturalproducts with improved properties obtained by any one of thetransformation methods described below. Plants transformed in accordancewith the present invention may be monocots or dicots and include, butare not limited to, rice, maize, wheat, barley, rye, sweet potato, sweetcorn, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli,turnip, radish, spinach, asparagus, onion, garlic, pepper, celery,squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum,cherry, peach, nectarine, apricot, strawberry, grape, raspberry,blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato,sorghum, sugarcane, sugar-beet, sunflower, rapeseed, clover, tobacco,carrot, cotton, alfalfa, potato, eggplant, cucumber, Arabidopsisthaliana, and woody plants such as coniferous and deciduous trees.Preferred plants to be transformed are rice, maize, wheat, barley,cabbage, cauliflower, pepper, squash, melon, soybean, tomato,sugar-beet, sunflower or cotton, but especially rice, maize, wheat,Sorghum bicolor, orchardgrass, sugar beet and soybean. The recombinantDNA sequences of the invention can be introduced into the plant cell bya number of well-known methods. Those skilled in the art will appreciatethat the choice of such method might depend on the type of plant whichis targeted for transformation, i.e., monocot or dicot. Suitable methodsof transforming plant cells include microinjection (Crossway et al.,1986, Bio Techniques 4:320–334), electroporation (Riggs and Bates, 1986,Proc. Natl. Acad. Sci., USA 83:5602–5606), Agrobacterium-mediatedtransformation (Hinchee et al., 1988, Bio/Technology 6:915–922; EP 0 853675), direct gene transfer (Paszkowski et al., 1984, EMBO J.3:2717–2722), and ballistic particle acceleration using devicesavailable from Agracetus, Inc., Madison, Wis. and Dupont, Inc.,Wilmington, Del. (see, for example, U.S. Pat. No. 4,945,050 and McCabeet al., 1988, Bio/Technology 6:923–926). The cells to be transformed maybe differentiated leaf cells, embryogenic cells, or any other type ofcell.

The invention will be further described by reference to the followingdetailed examples. These examples are provided for purposes ofillustration only, and are not intended to be limiting unless otherwisespecified.

EXAMPLES

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by J. Sambrook, et al.,Molecular Cloning: A Laboratory Manual, 3d Ed., Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press (2001); by T. J. Silhavy, M.L. Berman, and L. W. Enquist, Experiments with Gene Fusions, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M.et al., Current Protocols in Molecular Biology, New York, John Wiley andSons Inc., (1988), Reiter, et al., Methods in Arabidopsis Research,World Scientific Press (1992), and Schultz et al., Plant MolecularBiology Manual, Kluwer Academic Publishers (1998).

Example 1 Expression Vectors and Tranformation of Plants

Binary destination vectors for plant transformation consist of a binarybackbone and a T-DNA portion. The binary backbone contains the sequencesnecessary for selection and growth in Escherichia coli DH-5α(Invitrogen) and Agrobacterium tumefaciens LBA4404, including thebacterial spectinomycin antibiotic resistance aadA gene from E. colitransposon Tn7, origins of replication for E. coli (ColE1) and A.tumefaciens (VS1), and the A. tumefaciens virG gene. The T-DNA portionwas flanked by the right and left border sequences and includes thePositech™ (Syngenta) plant selectable marker and a gene expressioncassette which varies depending on the application. The Positech™ plantselectable marker in this instance consists of a rice ACT1 (actin)promoter driving expression of the PMI (phosphomannose isomerase) gene,followed by the cauliflower mosaic virus transcriptional terminator, andconfers resistance to mannose.

The gene expression cassette portion of the binary destination vectorsvaries depending on the application. In general, the cassette consistsof a promoter designed to express the gene in certain tissues of theplant, followed by cloning sites (in some cases interrupted by a segmentof spacer DNA), and finally by the A. tumefaciens nos 3′ endtranscriptional terminator. The promoters used are designed to expressthe gene of interest in specific target tissues (eg. endosperm: riceRS-4, wheat glutelin, maize ADPgpp or γ-zein, or barley α-thionin; eg.embryo: maize globulin or oleosin; eg. aleurone: barley α-amylase; eg.root: maize MSR1 and MRS3; eg. green tissue: maize PEPC) orconstitutively (eg. maize UBI plus intron), depending on the gene ofinterest. The cloning site contains either unique restriction enzymesites (for conventional cloning) and/or a Gateway™ recombination-basedcloning cassette (Invitrogen), in either the forward or reverseorientation. In gene expression cassettes designed for double-strandedinterfering RNA (dsRNA) production, the cloning site is divided by aspacer region (eg. first intron of the rice SH1 gene). The spacerpermits the cloning of two gene fragments one in the forward and one inthe reverse orientation. Antisense (reverse orientation expression) isanother technology available for silencing genes of interest.

Transformation of the nucleic acid molecules of the present inventioninto plants is performed using methods described above in the DetailedDescription.

Example 2 Abiotic Stress Tolerance cDNAs and Analysis

Table 1 lists candidate rice genes for biotic or abiotic stresstolerance described in this application.

TABLE 1 Abiotic Stress Tolerance Genes SEQ ID Putative Function &Similar Homology Reference Gene Nos: Genes and % Homology BOS1 1–2drought tolerance; dehydration induced bacterial infection myb-relatedproteins tolerance from the resurrection plant Craterostigmaplantagineum, Cpm7 (NCBI #T09737), Cpm5 (NCBI#T09736) and Cpm10(NCBI#T09735). These Cpm proteins show 76–80% identity to the BOS1protein over a stretch of the first 152 amino acids in the aminoterminus of the BOS1 protein

The abiotic stress tolerance genes are evaluated for their effect(s) intransformed plants by testing the transgenic transformed plants orprogeny plants as compared with non-transgenic plants. The plants aretested for their altered tolerance cold, drought, salt and heat usingmethods known to those skilled in the art, and examples of such assaysare described below.

Tolerance to salt is measured by using any salt tolerance assay known tothose skilled in the art. In particular, the salt tolerance assay isperformed essentially as follows:

Seeds from transformed plants and untransformed parental lines are sownon filter paper soaked with Yoshida solution placed in petri dishes.After 7 days of growth in the climate chamber seedlings (about 4 cmshoot length and 4 cm root length) will be exposed to salt stress asfollows: seedlings will be transferred to 24 well plates supplementedwith Yoshida solution (control) or Yoshida solution enriched with twodifferent salt concentrations (as below). To ensure the contact of theentire root with the solution a piece of moistened absorbent cotton isplaced on top of the root within the well flooded with the solution.Alternatively, the seeds may be grown in sand as a growth medium.

Control: Yoshida solution without supplementary salt

1) Yoshida solution enriched with NaCl 3.2 mg/l (48 mM)+CaCl₂ 3.6 mg/l(24 mM). This salt concentration evoked 50% growth reduction in shootlength.

2) Yoshida solution enriched with NaCl 6.4 mg/l (96 mM)+CaCl₂ 7.1 mg/l(48 mM).

Tissue is harvested at: 0, 6, 12, 24, and 36 hours. After exposure, theseedlings are separated into shoots and roots, or whole seedlings(whichever you prefer) and then immediately frozen in liquid nitrogenfor RNA extraction and analysis. Total RNA extraction is performed usingany known method in the art such as, an RNA extraction kit from Qiagen.

Alternatively, seedlings are observed for enhanced or decreasedtolerance to grown under salt stress as compared to the untransformedparental variety online. Samples are also taken for analysis of proteinexpression.

Yoshida Solutions

Stock Culture solution preparation (ml stock/ Final conc. Elem. Chemicalnames (g/10 L stock) 10 L solutions) (mM (ppm)) (Macro) N NH₄NO₃ 91412.5  1.43 mM (114 pm) P NaH₂PO₄.2H₂O 403 12.5  0.33 mM (51 ppm) K K₂SO₄714 12.5  0.51 mM (89 ppm) Ca CaCl₂ 886 12.5  1.0 mM (111 ppm) MgMgSO₄.7H₂O 3240 12.5  1.6 mM (394 ppm) (Micro)* Mn MnCl₂.4H₂O15–20 >0.01 mM Mo (NH₄)₆.MO₇O₂₄.4H₂O 1.5 1.5 × 10⁻⁴ mM B H₃BO₃ 12  0.02mM Zn ZnSO₄.7H₂O 0.7   3 × 10⁻⁴ mM Cu CuSO₄.5H₂O 0.31 1.6 × 10⁻⁴ mMCitric acid 119 12.5 0.071 mM of mixture (Iron)Fe Iron chelate 160  12.5 *Mix microelements in 10 L distilled water in the order above. pH,5.8 adjust with H₂SO₄ if necessary

For drought tolerance, substitute 20% polyethylene glycol (PEG; MW 8000)for the salt solution.

Example 3 BOS1 Identification and Isolation

Botrytis cinerea causes the gray mold diseases in horticultural cropsresulting in significant pre- and post-harvest loses. It is anecrotrophic fungal pathogen that infects a wide range of plant speciesin the field, greenhouse and storage. Very little is known about themolecular basis of the host response to B. cinerea and othernecrotrophic fungal pathogens. Although genetic variation for resistancehas been reported, no resistance gene has been identified. In search ofa genetic resistance mechanism, we initiated a screen to identify genesmediating resistance to Botrytis cinerea and other necrotrophicpathogens. We identified a botrytis susceptible (bos1) mutant from aT-DNA mutagenized population based on its increased susceptibility toBotrytis cinerea compared to wild type parental ecotype Col-0 plants(FIG. 1). The mutant was later tested and found to have increasedsusceptibility to another necrotrophic pathogen, Alternaria brassicicola(FIG. 2) and to a bacterial pathogen, Pseudomonas syringae (FIG. 3).Interestingly, the survival of bos1 plants was significantly reducedfollowing exposure to drought or salt stress. When grown in the presenceof 100 mM NaCl, only 18.75% of bos1 plants survived compared to 93.75%of wild type plants (FIG. 4). Similarly, when water was withheld fromsoil grown plants up to visible wilting, followed by watering, only 28%of the bos1 plants recovered, compared to 88% recovery in wild typeplants (FIG. 5). The mutant (bos1) was affected in the normal expressionof the gene encoding for a MYB transcription factor (referred to asAtMYB108 in the public data base, we refer to it as BOS1) due to a T-DNAinsertion (FIG. 6). In a preliminary assay, constitutive expression ofBOS1 (AtMYB108) under the regulation of the Arabidopsis UBQ3 promoterrestores the Botrytis susceptibility phenotype of the mutant to the wildtype level of resistance (FIG. 7). This confirmed that the mutation ofthe BOS1 (AtMYB108) gene is responsible for the bos1 mutant phenotype.These plants will be assayed for the complementation of the otherdiseases and abiotic stress response phenotypes of the bos1 mutant.

Plants resistant to one stress are often more resistant to others. Insome cases the resistance phenotypes could even transcend the biotic andabiotic stress boundary. This phenomenon, referred to as cross-tolerancecould be important for agriculture as crops could be developed totolerate more than one stress. Phenotypic data from the mutant suggeststhat the wild type BOS1 gene is involved in regulating responses to bothbiotic and abiotic stresses and a possibly in cross-regulation betweenresponses to microbial infection and responses to some abiotic stressfactors. Importantly, this makes the BOS1 gene a feasible candidate forgenerating crop varieties with increased tolerance to necrotrophicpathogens such as Botrytis cinerea, Alternaria spp, nd also fordeveloping crop varieties with increased tolerance to drought and saltstress.

Example 4 Cloning of the Gene Responsible for the bos1 Phenotype

The bos1 mutant was identified among transgenic Arabidopsis linesgenerated by transforming with Agrobacterium. We screened the T-DNAinsertion lines for botrytis susceptible (bos) mutants. The mutant wasfound in the family 7675 in a collection generated by SyngentaBiotechnology Inc. as susceptible to Botrytis cinerea compared to thewild type parental ecotype Colo-o plants. The Botrytis susceptibilityassay was done on 3-week old plants either by spraying a sporesuspension containing 100,000 spores per ml or drop inoculation of thespore suspension on leaves. Genetic and molecular analysis showed thatthe T-DNA insert co-segregates with the mutant phenotype. The mutant wasaffected in the normal expression of the MYB transcription factor due toa T-DNA insertion in the promoter region that leads to an alteredexpression (FIG. 6). In a preliminary assay constitutive expression ofthis gene under the regulation of the Arabidopsis UBQ3 promotercomplements or rescues the bos1 phenotype of the mutant (FIG. 7)

Expression analysis following Botrytis infection showed that BOS1 genewas induced following Botrytis infection (FIG. 8). However, diseaseinducibility of BOS1 was delayed and reduced in the coi1 mutant ofArabidopsis that is defective in JA perception (Xie et al., Science280:1091–4, 1998).

Genomic DNA from the mutant was isolated according to the proceduredescribed (Dellaporta et al., 1983) and used for TAIL PCR (Liu et al.,Plant J. 8(3):457–63, 1995) to amplify genomic fragment flanking theleft border of the T-DNA. TAIL PCR products contained sequence from themutagenizing T-DNA and adjacent Arabidopsis sequence. The T-DNA insertedin the promoter region of the BOS1 gene (SEQ ID NO:4).

BOS1 cDNA and Genomic Clones

The genomic sequence from TAIL PCR of bos1 mutant was used as the basisfor cloning the wild type BOS1 cDNA and genomic clones. The ends of thecDNA were determined by 5′and 3′RACE. Primers were designed based on thegenomic sequence obtained from the TAIL PCR product sequence. RACE wasperformed according to the SMART protocol from Clonetech. Poly A RNAfrom Col-0 wild type plants was reverse transcribed using oligo dTprimers provided by the SMART RACE cDNA Amplification Kit (Clontech).This was used as a template to amplify BOS1 cDNA using sense andantisense gene specific primers:

3′RACE Primer1: 5′-GACGTCCGCCGTGGAAACATTACACTT-3′ (SEQ ID NO:5) 3′RACEPrimer2: 5′-GGAAGAACGGACAACGAGATCAAGAAC-3′ (SEQ ID NO:6) 5′RACE Primer1:5′-TAGTACTCCGTTAAGTCTGACGCCGGAGA-3′ (SEQ ID NO:7) 5′RACE Primer2:5′-ATGCAAGATGACGTGCCGGCTGAT-3′ (SEQ ID NO:8)

Once the 5′and 3′ends of the BOS1 cDNA were determined by RACE, genespecific primers were designed to the start and stop codons and used toamplify a full-length cDNA using the following primers:

(SEQ ID NO:9) BOS1cDNA Forward primer: 5′-ATG GAT GAA AAA GGA AGA AGCTTG AAG-3′ (SEQ ID NO:10) BOS1cDNA Reverse primer: 5′-TCA GAA GCT ACCATT ATT GTT GAA CTG-3The BOS1 genomic region was cloned by using gene specific primersdesigned to include 1.5 Kb region upstream of the Start codon and areverse primer designed to the stop codon.

(SEQ ID NO:11) BOS1 Genomic Forward primer: 5′-TGC ACC AAA CCAA GTAA CAAGAGG-3′ (SEQ ID NO:12) BOS1 Genomic Reverse primer: 5′-CTAGCTAGCTCAGAAGCTACCATTATTGTT-3′

Example 5 Biotic Stress Assay

Fungal culture: Botrytis was grown on 2×V8 agar (36% V8 juice, 0.2%CaCO3, 2% Bacto-agar) at 20° C. Fungal cultures were initiated bytransferring pieces of agar containing mycelium to fresh 2×V8 agar andincubated at 20–25° C. Conidia were collected from 10 day old culturesby placing agar slices containing fungal material in 1% SabouraudMaltose Broth (SMB) buffer (DIFCO, Sparks, Md., USA, Becton Dickinson)and vortexing to release the spores. The suspension was passed throughmiracloth to separate the fungal material from pieces of agar.Botrytis assay: To infect plants, the fungal spore density was adjustedto 10⁵ spores/ml in SMB buffer and sprayed on four-five week old plantsusing a Preval sprayer. Single leaf inoculations were done by placing2–3 μL droplets of spore suspension of Botrytis on individual leaves offour-five week old plants. Following inoculation plants were kept undera transparent cover to maintain high humidity and transferred to agrowth chamber with 21° C. day and 18° C. night temperature and 12-hlight/12-h dark cycle. The light intensity was approximately 200μE/m²/s. Results of these assays were described above and in theFigures.

Example 6 BOS1 Promoter

The BOS1 promoter was cloned by amplifying the 1.5 KB genomic sequenceupstream of the translation start site of the BOS1 coding region (SEQ IDNO:1). This promoter fragment was fused to the GUS reporter gene for theanalysis of the regulation of the gene and pattern of expression. Thefollowing primers were used to clone the BOS1 promoter.

BOS1 Promoter Forward Primer: 5′-TCAATAGAAATCAGAAAACG-3′ (SEQ ID NO:13)BOS1 Promoter Reverse Primer: 5′-TGA TAT ACA CAG AAG AGA CCA-3′ (SEQ IDNO:14)

Example 7 Sequence Analysis

The Alignment of the BOS1 cDNA with the genomic sequence shows that theBOS1 gene is interrupted by 2 introns. The 1.2 Kb long cDNA of BOS1 (SEQID NO:1) contains an ORF capable of encoding a protein of 323 aminoacids (SEQ ID NO:2) with a predicted molecular mass of 37019.8 Da and atheoretical pl of 4.93. Insertion of the T-DNA in the bos1 mutantoccurred in the 5′UTR region. The BOS1 ORF encodes a putativeMYB-related protein with an ATP/GTP binding domain at the amino terminus(position 52–59 aa), followed by the two Myb DNA-binding domain repeatsignatures (position 24–32, 96–119 amino acids).

Sequence comparison reveals that the encoded BOS1 protein (SEQ ID NO:2)has significant sequence similarity to dehydration induced myb-relatedproteins from the resurrection plant Craterostigma plantagineum, Cpm7(NCBI #T09737), Cpm5 (NCBI#T09736) and Cpm10 (NCBI#T09735). These Cpmproteins show 76–80% identity to the BOS1 protein over a stretch of thefirst 152 amino acids in the amino terminus of the BOS1 protein.

Example 8 Complementation and Overexpression

The BOS1 cDNA (SEQ ID NO:1) was cloned into a binary vector andtransformed into bos1 and Col-0 wild type plants. Transgenic bos1 plantscarrying the UBQ3::BOS1cDNA were assayed for the Botrytis susceptibilityphenotype. Preliminary results show that these plants are comparable tonon-transgenic wild type plants in their level of Botrytis resistance(FIG. 7). Future testes will further evaluate disease and abiotic stresstolerance in UBQ3::BOS1 lines generated in bos1 and wild typebackgrounds. Data on diseases and abiotic stress tolerance of plantswith ectopic expression of the BOS1 gene will determine the utility ofthe BOS1 gene for plant improvement to diseases and stress tolerance.

Example 8 Construction of Binary Promoter::Reporter Plasmids

The entry vectors containing promoters of interest (the DNA sequence 5′of the initiation codon for the gene of interest) and resulting fromrecombination in a BP reaction between a PCR product using the promoterof interest as template and pDONRneo are used to construct a binarypromoter::reporter plasmid for Arabidopsis transformation. Theregulatory/promoter sequence is fused to the GUS reporter gene(Jefferson et al, 1987, EMBO J 6: 3901–3907) by recombination usingGATEWAY™ Technology according to manufacturers protocol as described inthe Instruction Manual (GATEWAY™ Cloning Technology, GIBCO BRL,Rockville, Md.). Briefly, according to this protocol the promoterfragment in the entry vector is recombined via the LR reaction with abinary Agrobacterium destination vector containing the GUS coding regionwith intron that has an attR site 5′ to the GUS reporter (pNOV2374). Theorientation of the inserted fragment is maintained by the att sequencesand the final construct is verified by sequencing. The construct is thentransformed into Agrobacterium tumefaciens strains by electroporation.

pNOV2374 is a binary vector with VS1 origin of replication, a copy ofthe Agrobacterium virG gene in the backbone and a Basta resistanceselectable marker cassette between the left and right border sequencesof the T-DNA. The Basta selectable marker cassette comprises theAgrobacterium tumefaciens manopine synthase promoter (AtMas, Barker, etal, Plant Mol. Biol. 2, 335–350 (1983)) operably linked to the geneencoding Basta resistance (denoted here as “BAR”, phosphinothricinacetyl transferase, White et al, Nucl Acids Res 18: 1062 (1990)) and the35S terminator. The AtMas promoter, BAR coding sequence and 35Sterminator are located at nt 4211 to 4679, nt 4680 to 5228, nt 5263 to5488 respectively, of pNOV2374. The vector contains GATEWAY™recombination components which were introduced into the binary vectorbackbone by ligating a blunt-ended cassette containing attR sites, ccdBand cholramphenicol resistance marker using the GATEWAY™ VectorConversion System (LifeTechnologies). The GATEWAY™ cassette is locatedbetween nt 126 and 1818 of pNOV2374. The promoter cassettes are insertedthrough an LR recombination reaction whereby the DNA sequence ofpNOV2374 between nt 126 and nt 1818 are removed and replaced with thepromoter of interest flanked by att sequences. The recombination resultsin the promoter sequence fused to the GUS reporter gene with intron(GIG) sequence. The GIG gene contains the ST-LS1 intron from Solanumtuberosum at nt 385 to nt 576 of GUS SEQ ID NO:2 (obtained from Dr.Stanton Gelvin, Purdue University, and described in Narasimhulu, et al1996, Plant Cell, 8: 873–886.). Shown below are the orientations of theselectable marker and promoter-reporter cassettes in the binary vectorconstructs.

For comparison of promoter activity an additional construct is producedwith the known Arabidopsis ubiquitin 3 (Ubq3(At), Callis, et al., J.Biol. Chem. 265: 12486–12493 (1990)) promoter plus intron operativelylinked to the GIG gene and the nos promoter. Shown below is theorientation of the selectable marker and promoter-reporter cassette inthe binary vector construct.

Binary Vector Construct:

-   -   RB Ubq3(At) promoter with intron fragment+GIG        gene+nos—AtMas+BAR+35S ter—LB

Example 10 Arabidopsis Transformation

1. Plant Preparation and Growth:

Arabidopsis seeds are sown on moistened Fafard Germinating Mix at adensity of 9 seeds per 4″ square pot, placed in a flat, covered with aplastic dome to retain moisture and moved to a growth chamber. Followinggermination the dome is removed and plants are grown for 3–5 weeks undershort days (8 hrs light) to encourage vegetative growth and productionof large plants with many flowers. Flowering is induced by providinglong days (16 hrs. light) for 2–3 weeks, at which time plants are readyfor dip inoculation into Agrobacterium to generate transgenic plants.2. Agrobacterium Transformation, Culture Growth and Preparation forPlant Infiltration:The binary promoter::reporter plasmids are introduced into Agrobacteriaby electroporation. The binary plasmid confers spectinomycin resistanceto the bacteria allowing cells containing the plasmid to be selected bygrowth of colonies on plates of LB+spectinomycin (50 mg/L). Presence ofthe correct promoter::GUS plasmid is confirmed by sequence analysis ofthe plasmid DNA isolated from the bacteria.Two days prior to plant transformation 5 mL cultures of LB+spectinomycin(50 mg/L) are inoculated with the Agrobacterium strain containing thebinary promoter::GUS plasmid and incubated at 30° C. for ˜24 hours. Each5 mL culture is then transferred to 500 mL of LB+spectinomycin (50 mg/L)and incubated for ˜24 hours at 30° C. Each 500 mL culture is transferredto a centrifuge bottle and centrifuged at 5000 rpm for 10 minutes in aSorvall Centrifuge. The supernatant is removed and the pelletedAgrobacterium cells are retained. The Agrobacterium cells areresuspended in 500 mL of modified Infiltration Media (IM+MOD: 50 g/Lsucrose, 10 mM MgCl, 10 uM benzylaminopurine) to which 50 ul of SilwetL-77 (Dupont) has been added.3. Plant Transformation by Dip Infiltration:Resuspended cells are poured into 1 L tri-pour beakers. Flowering plantsare inverted into the culture, making sure all inflorescences arecovered with the bacteria. The beakers are gently agitated for 30seconds, keeping all inflorescence tissue submerged. Plants are returnedto growth chamber following dip inoculation of the Agrobacterium. Asecond dip may be performed 5 days later to increase transformationfrequency. Seeds are harvested ˜4 to 6 weeks after transformation.4. Selection of Transgenic Arabidopsis:Seeds from transformed Arabidopsis plants are sown on moistened FafardGerminating Mix in a flat, covered with a dome to retain moisture andplaced in a growth chamber. Following germination seedlings are sprayedwith the herbicide BASTA. Transgenic plants are BASTA resistant due tothe presence of the BAR gene in the binary promoter::GUS plasmid.

Example 11 Reporter Gene Assays

Promoter activity is evaluated qualitatively and quantitatively usinghistochemical and florescence assays for expression of theβ-glucuronidase (GUS) enzyme.

1. Histochemical β-glucuronidase (GUS) Assay

For qualitative evaluation of promoter activity, various Arabidopsistissues and organs are used in GUS histochemical assays. Either wholeorgans or pieces of tissue are dipped into GUS staining solution. GUSstaining solution contains 1 mM 5-bromo4-chloro-3-indoly I glucuronide(X-Gluc, Duchefa, 20 mM stock in DMSO), 100 mM Na-phosphate buffer pH7.0, 10 mM EDTA pH 8.0, and 0.1% Triton X100. Tissue samples areincubated at 37° C. for 1–16 hours. If necessary samples can be clearedwith several washes of 70% EtOH to remove chlorophyll. Followingstaining tissues are viewed under a light microscope to evaluate theblue staining showing the GUS expression pattern.2. β-glucuronidase (GUS) Florescence AssayFor quantitative analysis of promoter activity in various Arabidopsistissues and organs, GUS expression is measured florometrically. Tissuesamples are harvested and ground in ice cold GUS extraction buffer (50mM Na2HPO4 pH 7.0, 5 mM DTT, 1 mM Na2EDTA, 0.1% TritonX100, 0.1%sarcosyl). Ground samples are spun in a microfuge at 10,000 rpm for 15minutes at 4° C. Following centrifugation the supernatant is removed forGUS assay and for protein concentration determination.To measure GUS activity the plant extract is assayed in GUS assay buffer(50 mM Na2HPO4 pH 7.0, 5 mM DTT, 1 mM Na2EDTA, 0.1% TritonX100, 0.1%sarcosyl, 1 mM 4-Methylumbelliferyl-beta-D-glucuronic acid dihydrate(MUG)), prewarmed to 37° C. Reactions are incubated and 100 uL aliquotsare removed at 10 minute intervals for 30 minutes to stop the reactionby adding to tubes containing 900 uL of 2% Na2CO3. The stopped reactionsare then read on a Tecan Spectroflourometer at 365 nm excitation and 455emission wavelengths. Protein concentrations are determined using theBCA assay following manufacturers protocol. GUS activity is expressed asrelative florometric units (RFU)/mg protein.

REFERENCES

Iturriaga G., Leyns L., Villegas A., Gharaibeh R (1996). “A family ofnovel myb-related genes from the resurrection plant Craterostigmaplantagineum are specifically expresses in callus and roots in responseto ABA or dessication”, Plant Molecular Biology 32: 707–716.

1. An isolated promoter comprising the nucleotide sequence of SEQ ID NO:4.
 2. The isolated promoter according to claim 1, wherein said promotercontrols abiotic stress responsive expression of an operably linkedcoding nucleotide sequence.
 3. An expression cassette comprising theisolated promoter according to claim
 1. 4. A recombinant vectorcomprising the expression cassette according to claim
 3. 5. A host cellstably transformed with the expression cassette of claim
 3. 6. The hostcell of claim 5, wherein said host cell is a plant cell.
 7. A plantcomprising the plant cell of claim
 6. 8. The plant of claim 7, whereinsaid plant is selected from the group consisting of maize, wheat,sorghum, rye, oats, turf grass, rice, barley, soybean, cotton, tobacco,sugar beet and oilseed rape.